IE912281A1 - New protein with cell-differentiation or cell-formation¹ability and recombinant process for the production - Google Patents

Abstract

A protein capable of causing differential growth of osteoblasts and cranial nerve cells originating in mouse calvarial cells, having 168 amino acid residues, and useful for treating and diagnosing osteoporosis and dementia. It is produced by expressing DNA which codes for this protein.

Description

Hoechst Japan Limited 90/S 008J Dr. KL SPECIFICATION Title of the invention New protein with cell-differentiation or cell-formation ability and recombinant process for the production Detailed Description of the Invention [Background of the invention] Field of industrial use The present invention relates to a novel protein having bone cell differentiation or. growth activity and brain cell differentiation or growth activity, a DNA coding said protein, and the production of said protein through gene technology.

Prior art Kadomatsu et al. (1988, Biochem. Biophys. Res. Commun., 151. 1312-1318) found a novel gene coding for a polypeptide with 9,971 dalton, named MK1, which is rich in basic lysine residues and is specifically expressed at the stage of early development of mouse embryonic teratocarcinoma. Kadomatsu et al. (1990, J. Cell Biol., 110, 607-616) also found that MK1 is a secreted polypeptide and a hormone for cell growth and differentiation like a protein belonging to the transforming growth factor (TGF) - beta family. Among proteins of TGFIE 912281 beta family, bone morphogenic proteins (BMPs) were recently discovered and were expected to be useful in the treatment of bone fracture (Wozney et al. 1988, Science, 242, 15281534). On the other hand, concerning brain cell derived growth factors, nerve growth factor (NGF) was also recently reported and was expected to be used in medical field.

Among the diseases for the aged persons, osteoporosis and dementia gradually become an big social issue in the advanced industrial countries. The number of patients suffering from these diseases are deemed to be increasing in future. Therefore, a gene specifically expressed in osteoblast cells or brain cells and its encoded product are expected to be useful for diagnosis and/or treatment of these diseases.

[An outline of the invention] The present invention provides a cDNA clone specifically expressed in an osteoblastic cell line, MC3T3E1, established from mouse calvaria, and also a novel protein coded by the cDNA. The present invention also provides a cDNA clone coding the corresponding human protein isolated from a cDNA library constructed from a human cell using a probe of cDNA coding the novel mouse protein.

The present invention also provides a protein which has 168 amino acids residues. The novel protein belongs to the MK-family which is a group of proteins with celldifferentiation or cell-growth activity. The mRNA coding the protein is strongly expressed in MC3T3E1, and weakly in brain cells and NIH3T3 cells, but not in the mouse kidney, spleen, thymus, liver, lung or skeletal muscle.

First, the invention discloses a protein having the amino acid sequence of the following formula [I].

Met-A*l-A*2-Gln-Gln-Tyr-Gln-Gln-Gln-Arg-Arg-Lya-Phe-Ala-AlaAla-Phe-Leu-Ala-A*3-Ile-Phe-Ile-Leu-Ala-Ala-Val-Asp-Thr-AlaGlu-Ala-Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lya-Lys-Val-Lya-Lys-SerAsp-Cya-Gly-Glu-Trp-Gln-Jrp-Ser-Val-Cys-Val-PrQ-Thr-Ser-GlyAap-Cys-Gly-Leu-Gly-Thr-Arg-Glu-Gly-Thr-Arg-Thr-Gly-Ala-GluCys-Lys-Gln-Thr-Met-Lya-Thr-Gln-Arg-Cys-Lys-Xle-Pro-Cya-AsnTrp-Lys-Lys-Gln-Phe-Gly-Ala-Glu-Cys-Lya-Tyr-Gln-Phe-Gln-AlaTrp-Gly-Glu-Cys-Asp-Leu-Asn-Thr-Ala-Leu-Lya-Thr-Arg-Thr-GlySer-Leu-Lys-Axg-Ala-Leu-Hia-Aan-Ala-A*4-Cys-Gln-X,ya-Thr-ValThr-Ile-Ser-Lys-Pro-Cys-Gly-Lya-Leu-Thr-Lya-Pro-Lys-Pro-GlnAla-Glu-Ser-Lya-Lya-Lys-Lya-Lys-Glu-Gly-Lys-Lya-Gln-Glu-LysMet-X^u-Asp [I] In this amino acid sequence, Α·1 is Ser or Gin, A«2 is Ser or Ala, A*3 is Leu or Phe, and A»4 is Asp or Glu.

A desirable example of proteins provided by the invention is a protein with an amino acid sequence described in the following formula [I-I] of mouse OSF-1 or [I-IIJ of human OSF-1.

Met-Ser-Ser-Gln-Gln-Tyr-Gln-Gln-Gln-Arg-Arg-Lys-Phe-Ala-AlaAla-Phe-Leu-Ala-Leu-Ile-Phe-Ile-Leu-Ala-Ala-Val-Aap-Thr-AlaGlu-Ala-Gly-Lys-Lya-Glu-Lys-Pro-Glu-Lys-Lya-Val-Lys-Iiys-SerAap-Cys-Gly-Glu-Trp-Gln-Trp-Ser-Val-Cys-Val-Pro-Thr-Ser-GlyAap-Cya-Gly-Leu-Gly-Thr-Arg-Glu-Gly-Thr-Arg-Thr-Gly-Ala-GluCys-Lya-Gln-Thr-Met-Lys-Thr-Gln-Arg-Cys-Lys-Ile-Pro-Cya-AsnTrp-Lya-Lya-Gln-Phe-Gly-Ala-Glu-Cys-Lya-Tyr-Gln-Phe-Gln-AlaTrp-Gly-Glu-Cya-Aap-Leu-Aan-Thr-Ala-Leu-Lys-Thr-Arg-Thr-GlySer-Leu-Lya-Arg-Ala-Leu-His-Asn-Ala-Asp-Cys-Gln-Lya-Thr-ValThr-Ile-Ser-Lys-Pro-Cys-Gly-Lys-Leu-Thr-Lys-Pro-Iiys-Pro-GlnAla-Glu-Ser-Lya-Lya-Lys-Lys-Lys-Glu-Gly-Lya-Lys-Gln-Glu-LysMet-Leu-Asp [I-I] Met-Gln-AJ.a-Gln-Gln-Tyr-Gln-Gln-Gln-Arg-Arg-Lya-Phe-Ala-AlaAla-Phe-Leu-Ala-Phe-Ile-Phe-Xle-Leu-Ala-Ala-Val-Asp-Thr-AlaGlu-Ala-Gly-Lya-Lya-Glu-I The invention also discloses a DNA coding the amino acid sequence in formula [I].

A portion of the amino acid residues in formula [I] can be modified by deletion, substitution or addition of amino acid residues to the extent that the activity of the protein is not reduced. In addition, not only DNAs coding the fulllength amino acid sequence of formula [I] or its partially modified sequences, but also a portion of the DNA is included in the invention.

The invention also concerns an expression vector comprising 1) DNA coding the protein to be produced, 2) a suitable promoter, such as SV 40 virus T antigen early promoter, rightly oriented upstream of the coding sequence and 3) the other DNA fragments necessary to express an exogenous gene in the host cell.

The invention also concerns a method for the production of the proteins with the amino acid sequence of formula [I], comprising 1) transformation of a host cell with the expression vector provided by the invention, 2) cultivation of the transformed cells, then 3) production of the said protein.

The invention also concerns a protein obtained by the cultivation of the host cells transformed by the expression vector provided by the present invention. A protein means 1) a protein with the full-length amino acid sequence of formula [I], or 2) a protein with full-length, but modified amino acid sequences obtained through deletion, substitution or addition of amino acids to the sequence of formula [I], or 3) a portion of the amino acid sequences of 1) or 2).

The effect of the invention The DNA coding 168 amino acid residues provided by the invention can be used in the production of a protein with the full-length 168 amino acid sequence or Its fragments.

The said DNA can be ligated with any suitable promoter such as SV40 virus T antigen early promoter and inserted into an expression vector. The said protein can be secreted by the host cell transformed by the said expression vector. Since the protein has no glycosylation site in its amino acid sequence, the protein can be produced using bacteria such as E. coll, or yeasts. The protein produced by the invention has the cell-differentiation or cell-growth activity in the same manner as the other proteins belonging to the MK family. Therefore the protein is expected to be useful in the treatment and/or diagnosis of osteoporosis and dementia. The said DNA can be used as a probe for the Isolation of human counterparts from the cDNA library constructed from an established cell line from human osteoblastoma or brain neuron cells or its tumor cells.

[Embodied description of the Invention] OSF-1 Protein The protein provided by the invention was picked up in a cDNA library of MC3T3E1 cells, an osteoblastic cell line established from the mouse calvaria, through a differential hybridization screening compared with NIH3T3, an established fibroblast cell line. Then, using the cDNA coding mouse OSF-1 as a probe, a cDNA coding human OSF-1 was selected from the human cDNA library. This nucleotide sequence coding the said protein is disclosed in the formula [II].

The tissue specificity of mouse OSF-1 was examined with the Northern blotting method among the several mouse tissues such as brain, muscle, kidney, liver, spleen, thymus and lung. The positive signals, which means the expression of mRNA coding the said protein, was observed only in brain.

OSF-1 has the following characteristic profile: (1) It contains 168 amino acids residues including the first methionine at the start site, and it is a highly basic and hydrophilic protein containing 17%-18% lysine residues out of the total of the amino acid residues. (2) It contains no glycosylation site. (3) It contains ten cysteine residues, which suggests the highly complex three dimensional structure of OSF-1. (4) The profile of OSF-1, mentioned in (1) - (3), suggested the high homology between OSF-1 and MK1 which relates to the cell-growth and differentiation. Based on the each amino acid sequences, the sequential homology between MK1 and mouse OSF-1 is calculated as 48.3 %. The amino acid residues with homology (designated as ·) are shown in the following scheme.

OSF-1 1 50 EAGKKEKPEK KVKKSDCGEW MSSQQYQQQR RKFAAAFLAL IFILAAVDTA 51 100 OSF-1 QWSVCVPTSG DCGLGTREGT RTGAECKQTM KTQRCKIPCN WKKQFGAECK A A A A AA AAA ★ Ait* t* MK-1 MGF REGT-CGAQT QRVHCKVPCN WKKEFGADCK 1 32 101 150 OSF-1 YQFQAWGECD LNTALKTRTG SLKRALHNAD CQKTVTISKP CGKLTKPKPQ * * aa aa A A * * AA A A AA A AA A AAA MK-1 YKFESWGACD GSTGTKARQG TLKKARYTAQ CQETIRVTKP CTSKTKSKTK 33 82 151 168 OSF-1 AESKKKKKEG KKQEKMLD * * * MK-1 AKKGKGKD 90 MK1 is a secreted protein (or polypeptide) and is deemed to be a differentiation factor of embryonic carcinoma cells controlled by the retinoic acid (M. Tomomura et al., Seikagaku, 1989, 61, 1040). OSF-1 is very similar to MK1 except for its tissue specificity to the osteoblastoma and brain. The protein provided by the Invention includes the full-length amino acid sequence of OSF-1, or a fragment of OSF-1 with substantially the same activity of OSF-1.

The protein of the invention can be secreted as a portion of the amino acid sequence in formula [I]. Based on the homology of amino acid sequence of each protein, OSF-1 is likely to be cleaved at the 78th Gin residue and the 84th Arg residue, then to be secreted as a protein of about 9,000 dalton. Therefore the protein of the Invention includes parts of the protein of formula [I] and the mutants of OSF1 with substitution of a few amino acid residues with substantially the same activity of OSF-1.

DNA coding OSF-1 protein DNA coding the protein of the present Invention means 1) a DNA having a nucleotide sequence coding the amino acid sequence of formula [I], or 2) a DNA coding either a portion of OSF-1 or substitutes of amino acid residues with substantially the same activity of OSF-1.

A typical nucleotide sequence is disclosed in the following formula [II].

ATG-YZG-XCY-CAU-CAU-TAY-CAG-CAG-CAU-CGT-ZGA-AAA-TTT-GCA-GCTGCC-TTC-YTG-GCA-TTV-ATT-TTC-ATZ-YTG-GCA-GCT-GTG-GAY-ACT-GCTGAU-GCZ-GGG-AAG-AAA-GAG-AAA-CCW-GAA-AAA-AAU-GTG-AAU-AAG-TCTgac-tgt-gga-gaa-tgg-cag-tgg-agt-gtg-tgy-gtg-ccy-acc-agy-gguGAC-TGT-GGU-YTG-GGC-ACZ-CGG-GAG-GGC-ACT-CGV-ACT-GGZ-GCY-GAGTGC-AAU-CAU-ACC-ATG-AAG-ACY-CAG-AGA-TGT-AAG-ATC-CCY-TGC-AACTGG-AAG-AAG-CAU-TTT-GGZ-GCX-GAG-TGC-AAU-TAC-CAG-TTC-CAG-GCYTGG-GGA-GAA-TGT-GAC-CTV-AAY-ACZ-GCC-YTG-AAG-ACC-AGA-ACT-GGZAGY-CTG-AAG-CGA-GCY-CTG-CAC-AAT-GCY-GAZ-TGY-CAG-AAV-ACT-GTCACC-ATC-TCC-AAG-CCC-TGT-GGC-AAU-CTV-ACC-AAG-CCC-AAU-CCT-CAAGCU-GAU-TCW-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAGATG-CTG-GAT-TAA [II] In this nucleotide sequence, U is A or G, Y is C or T, X is G or T, Z is A or C, V is G or C and W is T or A.

Examples for an embodiment of DNA coding mouse OSF-1 and human OSF-1 are described in the following formula [III] and [II-II], respectively.

ATG-TCG-TCC-CAG-CAA-TAT-CAG-CAG-CAA-CGT-AGA-AAA-TTT-GCA-GCTGCC-TTC-CTG-GCA-TTG-ATT-TTC-ATC-TTG-GCA-GCT-GTG-GAC-ACT-GCTGAG-GCC-GGG - AAG-AAA-GAG-AAA- CCT -GAA-AKA-AAG-GTG-AAA-AAG - TCT GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGC-GTG-CCT-ACC-AGC-GGGGAC-TGT-GGA-TTG-GGC-ACC-CGG-GAG-GGC-ACT-CGC-ACT-GGC-GCC-GAGTGC-AAA-CAG-ACC-ATG-AKG-ACT-CAG-AGA-TGT-AAG-ATC-CCT-TGC-AACTGG-AAG-AAG-CAG-TTT-GGA-GCT-GAG-TGC-AAG-TAC-CAG-TTC-CAG-GCTTGG-GGA-GAA-TGT-GAC-CTC-AAT-ACC-GCC-TTG-AAG-ACC-AGA-ACT-GGCAGC-CTG-AAG-CGA-GCT-CTG-CAC-AAT-GCT-GAC-TGT-CAG-AAA-ACT-GTCACC-ATC-TCC-AAG-CCC-TGT-GGC-AAG-CTC-ACC-KAG-CCC-AAG-CCT-CAAGCG-GAG-TCA-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAGATG-CTG-GAT-TAA [II-I] ATG-CAG-GCT-CAA-CAG-TAC-CAG-CAG-CAG-CGT-CGA-AAA-TTT-GCA-GCT GCC-TTC-TTG-GCA-TTC-ATT-TTC-ATA-CTG-GCA-GCT-GTG-GAT-ACT-GCT GAA-GCA-GGG-AAG-AAA-GAG-AAA-CCA-GAA-AAA-AAA-GTG-AAG-AAG-TCT GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGT-GTG-CCC-ACC-AGT-GGA GAC-TGT-GGG-CTG-GGC-ACA-CGG-GAG-GGC-ACT-CGG-ACT-GGA-GCT-GAG TGC-AAG-CAA-ACC-ATG-AAG-ACC-CAG-AGA-TGT-AAG-ATC-CCC-TGC-AAC TGG-AAG-AAG-CAA-TTT-GGC-GCG-GAG-TGC-AAA-TAC-CAG-TTC-CAG-GCC TGG-GGA-GAA-TGT-GAC-CTG-AAC-ACA-GCC-CTG-AAG-ACC-AGA-ACT-GGA AGT-CTG-AAG-CGA-GCC-CTG-CAC-AKT-GCC-GAA-TGC-CAG-AAG-ACT-GTC ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAA-CTG-ACC-AAG-CCC-AAA-CCT-CAA GCA-GAA-TCT-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG ATG-CTG-GAT-TAA III-II] Based on the amino acid sequences, the nucleotide sequences coding the amino acid sequence can be determined using generally available genetic codon tables. Therefore, the DNA coding for the protein provided by the Invention is either the DNA with the nucleotide sequence of formula [II] or its degeneracy Isomer. Degeneracy isomer means a DNA having a nucleotide sequence coding the identical protein with OSF-1 but usaglng equivalent codon(s).

Preparation of DNA coding OSF-1 protein The DNAs of the present invention can be synthesized by any standard method for chemical synthesis of oligonucleotides based on the nucleotide sequences disclosed in this invention.

The DNA of the invention coding mouse OSF-1 can be also prepared with the following typical procedures of gene technology, that is 1) seeding of 2.2 x 10s per plate (plastic culture plate, 10 cm diameter, total 40 plates) of an osteoblastic cell line, MC3T3E1 established from mouse calvaria (Kodama et al., Jpn. J. Oral. Biol. 23, 899-901), 2) cultivation of the cells for about 9 days at 37 °C with optionally changing the medium, 3) harvesting the cells when the number of the cells reaches 8 x 10T, 4) isolation of total RNA from the cells, 5) purification of the mRNA, 6) preparation of a cDNA library, then 7) cloning of the desired cDNA. References for steps 4) - 7) are made to the published manuals such as Molecular Cloning: A Laboratory Manual (1982, edited by T. Maniatis et al. and published by Cold Spring Harbor Laboratory).

It is also possible to isolate cDNA of human or species other than mouse from a cDNA library of the corresponding species using the mouse cDNA as a probe for screening. Expression of OSF-1 protein OSF-1 can be produced by any host cell, such as E. coll, yeast or animal cells with the suitable promoter for each cell. The detailed procedures can be performed according to the methods described in the Maniatis's manual (ibid.) etc.

The present invention is explained using preferred embodiments. The methods used in the preferred embodiments are as follows.

Restriction enzymes were purchased from New England Biolabs (U.S.A.) and Takara Shuzo (Japan), and used according to the supplier’s instruction. Deletion kit, T4 ligase and bacterial alkaline phosphatase were purchased from Takara Shuzo. cDNA synthesis system plus was purchased from Amersham (England). AMV reverse transcriptase, mRNA purification kit and EcoRI-Notl adaptor were purchased from Pharmacia (Sweden). Random primed DNA labeling kit was purchased from Boehringer Mannheim Yamanouchi (Japan). Lambda gtlO cloning vector and in vitro packaging kit Glgapack gold were purchased from Stratagene (U.S.A). Transformation of E. coli and DNA ligation were performed as described by Maniatis et al (ibid.).

Exp. 1 Preparation of a cDNA library from murine osteoblastic cell line MC3T3E1 cells Total RNA was extracted by the guanidine method (Maniatis et al., ibid.) from 8 x 107 cells of the murine osteoblastic cell line, MC3T3E1, then mRNA was purified from total RNA by the mRNA purification kit (Pharmacia). Double strand cDNA was synthesized from the mRNA by cDNA synthesis system plus. The synthesized double strand cDNA was cloned into the lambda gtlO cloning vector after the ligation of an EcoRI-Notl adaptor by T4 DNA ligase, and packaged Into lambda particles by the in vitro packaging kit Gigapack gold. Packaged phages were stored in SM buffer (Maniatis et al. ibid.). The efficiency of this library was estimated by infecting E. coll C600 (Japanese Cancer Research Resources Bank. National Institute of Health of Japan, HT003) with these phages, resulting in 1.4 x 107 plaques/ug cDNA.

Exp. 2 Picking up of MC3T3E1 specific clones selected by differential screening against mouse fibroblastic cell line NIH3T3 and recloning Into plasmid vector pUC118 Radioactive cDNA probe was synthesized using AMV reverse transcriptase from the mRNA of either MC3T3E1 cells or NIH3T3 cells (ATCC CRL 1658) prepared according to the same method as MC3T3E1 cells. About 1.5 x 104 clones of the cDNA library of MC3T3E1 cells prepared in Exp. 1 were screened by plaque hybridization using each radioactive cDNA probe one by one, and 78 clones which hybridize specifically to the cDNA probe of MC3T3E1 were picked up.

Phage DNA of each clone was extracted with phenol and precipitated by 70 % ethanol (Maniatis et al. ibid.), and the cDNA insert of each clone was purified after size fractionation by agarose gel electrophoresis of EcoRI digested phage DNA (Maniatis et al., ibid.). Radioactive probes of each clone were prepared from them using the random primed DNA labeling kit (Boehringer Mannheim Yamanouchi). 0.3 ug of mRNA prepared from either MC3T3E1 or NIH3T3 was subjected to formaldehyde denaturing agarose gel electrophoresis and transferred to a Nylon membrane (BYODYNE, Pall Bio Support, U.S.A.) by capillary blotting (Maniatis et al., ibid.).

Each clone was further screened for its expression rate in MC3T3E1 cells and NIH3T3 cells by the Northern blotting method with a radioactive probe derived from each clone.

Then one clone, named MC031, was selected for its specific expression in MC3T3E1 cells.

The cDNA insert which was obtained by digestion of phage clone MC031 with EcoRI was cloned into cloning vector pUC118 (Takara Shuzo) while eliminating its 5* terminal phosphate by microbial alkaline phosphatase (Takara Shuzo), by means of T4 DNA ligase to produce plasmid pMC031, the structure of which is shown in Fig. 1.

Exp. 3 DNA sequencing of cDNA insert of pMC031 After the digestion of pMC031 with SphI and BamHI, 10 deletion mutants were constructed using the kilo sequence deletion kit (Takara Shuzo). These deletion mutants were designated as pM031-l, pMC031-2, ... and pMC031-10 (Fig. 2) The DNA sequence of the cDNA Insert of pMC031 and Its deletion mutants were determined using an automatic DNA sequencer (model 370A, Applied Blosystems), in the orientation from the Hindlll site to the EcoRI site.

Approximately 300 base pairs of the nucleotide sequence of each clone were determined, and the entire sequence of the cDNA was composed by combining the overlapping sequence data. The entire sequence and its deduced amino acid sequence are shown in Fig. 3, and the protein coded by this cDNA was designated as mouse OSF-1.

Exp. 4 Tissue specific expression of mouse OSF-1 Northern blotting was performed to examine the tissue specific expression of mouse OSF-1.

Total RNA was prepared by the guanidine method from thymus, spleen, brain, kidney, lung, liver, and skeletal muscle, which were taken out from ten 4-weeks-old mice (C57BL/6N) purchased from CLEA JAPAN Inc.. Ten ug of RNA prepared from each tissue, MC3T3E1 or NIH3T3 was subjected to formaldehyde denaturing agarose gel electrophoresis, and fixed on nylon membranes. pMC031 was digested with EcoRI and the cDNA insert fragment was size fractionated by agarose gel electrophoresis and purified. A radioactive probe was synthesized from this fragment by the random primed DNA labeling kit.

Northern blot analysis was performed using these membranes and probe, and high expression was observed in MC3T3E1 and rather low expression was observed in brain and NIH3T3 (Fig. 4).

Exp. 5 Cloning of a cDNA coding human OSF-1 Using pMC031 containing cDNA coding mouse OSF-1 as a probe, 4 x 104 clones of a cDNA library for human cerebral superior temporal gyrus (Clonetech Co., code No. CLHL1094a) were screened by plaque hybridization, then one positive phage clone was picked up. The clone was named as HBR1.

Then the cDNA insert in HBR1 was isolated by EcoRI digestion and ligated to pUC118 (Takara Shuzo Co., Japan) to create pHBRl, corresponding to pMC031.

Exp. 6 DNA sequencing of cDNA insert of pHBRl After the digestion of pHBRl with Pstl and BamHI, 10 deletion mutants were constructed using the kilo sequence deletion kit (Takara Shuzo). These deletion mutants were designated as pHBRl-Ι, pHBRl-2, pHBRl-3, ... and pHBRl-10.

The DNA sequences of the cDNA insert of pHBRl and its deletion mutants were determined using an automatic DNA sequencer (model 370A, Applied Biosystems), in the orientation from the Hindlll site. Approximately 200 base pairs of nucleotide sequence of each clone were determined, and the entire sequence of the cDNA was composed by combining the overlapping sequence data. The entire sequence and its deduced amino acid sequence are shown in Fig. 6.

Exp. 7 Construction of an expression vector for OSF-1 and production of OSF-1 using this vector The expression of OSF-1 was performed according to the method disclosed In Japanese Laid-open Sho63-267288 titled The method for expressing proteins in animal cells using in vitro amplified genes”.

The cDNA for OSF-1 was inserted into cassette vector PHSG747 (Japanese Cancer Research Resources Bank. National Institute of Health of Japan, VE047), between the SV40 T antigen early promoter and the poly A additional signal, as described below.

Firstly, the EcoRI site upstream of the SV40 T antigen early promoter of pHSG747 was removed by gap filling using the Klenow fragment (Takara Shuzo) and followed by religation with ligase. An unique new EcoRI site was Introduced at the unique Pstl site between the SV40 promoter and the poly A signal by inserting a synthetic EcoRI linker oligonucleotide. The newly constructed vector, designated as pHSG757, was used for in vitro gene amplification (Fig. 5).

The cDNA fragment of OSF-1 was inserted into the EcoRI site of pHSG757, in the right orientation for the expression of OSF-1, and the plasmid was designated as pOFl. An OSF-1 expression unit fragment having asymmetric cohesive ends [III] can be obtained by digesting pOFl with BstXI.

’ CTGG CCACGGGG 3' OSF-1 Expression Unit 3’ CCCCGACC GGTG 5’ [HI] However, the digestion of pOFl with BstXI generates two fragments very close in size, and it is rather difficult to separate these two fragments by size fractionation. Therefore, the plasmid pHSGll (Japanese Cancer Research Resources Bank, National Institute of Health of Japan, VE049) was inserted at the Xhol site downstream of the poly A signal of pOFl. The plasmid constructed was designated as pOF3.

An expression fragment [III] mentioned above was mixed with DNA fragment [IV] prepared from cosmid vector pHSG293 (Japanese Cancer Research Resources Bank, National Institute of Health of Japan, VE046) at a molecular ratio of 20:1 then ligated tandemly by T4 ligase as disclosed in Japanese Laidopen Sho63-267288.

’ CTGG neo gene + cos site CCACGGGG 3* 3’ CCCCGACC [IV] GGTG 5’ The long chain DNA fragment prepared was packaged Into lambda phage in vitro and amplified In E. coli 0m206 (deposited to Fermentation Research Institute, Agency of Industrial Science and Technology, Tsukuba, as FERM P-9110; Takeshita et al. 1988, Gene, 71, 9-18) and packaged in vivo into lambda phage particles to prepare a large amount of the cosmid DNA. The DNA was transfected into CHO (Chinese Hamster Ovary) cells by the conventional calcium phosphate co-precipitation method and G418 resistant clones were selected. Then the total RNA of each clone was prepared by the guanidine method and the expression level of the OSF-1 gene was determined by Northern blot analysis using OSF-1 cDNA as probe, and the highest producer clone was selected.

The cells of the selected clone were cultured in alphaMEM containing 10 % fetal calf serum for 24 hours, and the OSF-1 protein was detected in the culture supernatant.

Brief description of drawings Figure 1 shows a plasmid map of pMC031. The open box is the inserted cDNA for mouse OSF-1. The arrow is the ampicillin resistant gene. The closed circle is the replication origin of the plasmid.

Figure 2 shows a structure of the cDNA insert segments of pMC031 and its 10 deletion mutants. Solid lines are inserted cDNAs contained in each plasmid. Arrows are regions whose sequence was determined and the direction of nucleotide sequencing.

Figure 3 shows the entire nucleotide sequence of the inserted cDNA of pMC031, and the amino acid sequence of mouse OSF-1 deduced from the nucleotide sequence.

Underlined is the EcoRI-Notl linker segment. A symbol with three asterisks, ·*·, is the translation termination codon.

Figure 4 shows the result of the Northern blotting analysis for tissue specificity of OSF-1 gene expression. Each lane indicates: 1; thymus, 2; spleen, 3; brain, 4; kidney, 5; liver, 6; lung, 7; skeletal muscle, 8; MC3T3E1 cells, and 9; NIH3T3 cells. 18S and 28S Indicate the positions of ribosomal RNAs.

Figure 5 shows a plasmid map of pHSG757. The open box is the poly A signal. The open arrow is the SV40 T antigen early promoter. The arrow is the direction and approximate location of the chloramphenicol resistance gene. The closed circle is the replication origin of the plasmid.

Figure 6 shows the entire nucleotide sequence of the inserted cDNA of pHBRl, and the amino acid sequence of human OSF-1 deduced from the nucleotide sequence.

Claims (16)

HOE 90/S 008J Claims

1. ) A protein with the amino acid sequence of the following formula [I], or with an amino acid sequence of a partially modified formula Met-A*1-A*2-Gln-Gln-Tyr-Gln-GlnAla-Phe-Leu-Ala-A*3-Xle-Ph«-Il*· Glu-Ala-Gly-Lys-Lya-Glu-Lya-Pxo' Aap-Cya-Gly-Glu-Txp-Gln-txp-SexAap-Cya-Gly-Leu-Gly-Thx-Arg-GluCya-Lya-Gln-Thx-Met-Lya-Thx-Gln· Txp-Lya-Lys-Gln-Phe-Gly-Ala-Glu· Txp-Gly-Glu-Cya-Asp-Leu-Aan-Thx· Sex-Leu-Lya-Axg-Ala-Leu-Hia-Aan Thr-Ile-Sex-Lya-Pxo-Cya-Gly-Lya Ala-Glu-Sex-Lya-Lya-Lya-Lya-Lya Met-Leu-Asp [I] [I], or their parts Gln-Axg-Axg-tya-Phe-Ala-AlaLeu-Kla-Ala-Val-Asp-Thr-AiaGlu-Lya-Lya-Val-Lya-Lya-SerVal-Cya-Val-Pxo-Thx-Sex-GlyGly-Thx-Axg-Thx-Gly-Ala-GluAxg-Cya-Lya-Xle-Pro-Cya-Asn-Cys-Lya-Tyx-Gln-Phe-Gln-Ala-AJ.a-Leu-Lya-Thr-Axg-Thx-Gly-Ala-AM-Cya-Gln-Lya-Thx-Val-Leu-Thr-Lya-Pro-Lya-Pxo-Gln-Glu-Gly-Lys-Lya-Gln-Glu-Lyawherein A*1 is Ser or Gin, A«2 is Ser or Ala, A«3 is Leu or Phe, and A*4 is Asp or Glu.

2. ) A protein according to claim 1 with the amino acid sequence of the following formula II-I] Mat-Sex-Sex-Gln-Gln-Tyx-Gln-Gln-Gln-Arg-Axg-Lya-Pha-Ala-AlaAla-Phe-Leu-Ala-Leu-Ile-Phe-Ile-Leu-Ala-Ala-Val-Aap-Thx-AlaGlu-Ala-Gly-X.ya-I«y8-Glu-Lya-Fxo-Glu-I.ya-Xya-Val-Lye-I.y8-SaxAsp-Cya-Gly-Glu-Txp-Gln-Txp-Sax-Val-Cya-Val-Pxo-Thx-Sax-GlyAsp-Cys-Gly-Leu-Gly-Thr-Arg-Glu-Gly-Thr-Axg-Thr-Gly-Ala-GluCya-Lya-Gln-Thx-Met-Lya-Thx-Gln-Axg-Cya-Lya-Ila-Pxo-Cys-AanTrp-Lya-Lya-Gln-Phe-Gly-Ala-Glu-Cya-Lya-Tyr-Gln-Phe-Glu-AlaTrp-Gly-Glu-Cya-Aap-Leu-Aan-Thx-Ala-Leu-Lya-Thx-Arg-Thx-GlySex-Leu-Lya-Axg-Ala-Leu-His-Aan-Ala-Aap-Cya-Gln-Lya-Thx-ValThx-Ile-Sex-Lys-Pxo-Cya-Gly-Lya-Lau-Thx-Lya-Pro-Lya-Pro-GlnAla-Glu-Sex-Lya-Lys-Lya-Lya-I.y8-Glu-Gly-I.y8-X.ya-Gln-Glu-I

3. ) A protein according to claim 1 with the amino acid sequence of the following formula [I-II] Met-Gln-Ala-Gln-Gln-Tyx-Gln-Gln-Gln-Arg-Axg-Lys-Phe-Ala-Ala Ala-Phe-Leu-Ala-Phe-Ile-Phe-Ile-Leu-Ala-Ala-Val-Asp-Thx-Ala Glu-Ala-Gly-Lys-Lys-Glu-Lys-Pxo-Glu-Xys-I.ys-Val-Lys-Lys-Sex Asp-Cys-Gly-Glu-Txp-Gln-Txp-Sex-Val-Cya-Val-Pxo-Thx-Sex-Gly Aap-Cys-Gly-Leu-Gly-Thx-Axg-Glu-Gly-Thx-Arg-Thr-Gly-Ala-Glu Cya-Lys-Gln-Thr-Met-Lys-Thr-Gln-Arg-Cys-Lys-Ile-Pro-Cys-Asn Trp-Lya-Lya-Gln-Phe-Gly-Ala-Glu-Cya-Lya-Tyx-Gln-Phe-Gln-Ala Txp-Gly-Glu-Cya-A3p-Leu-Aan-Thx-Ala-Leu-X.ya-Thx-Axg-Thx-Gly Ser-Leu-Lya-Axg-Ala-Leu-Hia-Aan-Ala-Glu-Cya-Gln-Lya-Thx-Val Thx-Ile-Sex-Lya-Pxo-Cya-Gly-Lya-Leu-Thx-lya-Pro-Lya-Pxo-Gln Ala-Glu-Ser-Lya-Lya-Lya-Lya-Lya-Glu-Gly-Lya-Lya-Gln-Glu-Lya Met-Leu-Aap il-II]

4. ) A DNA coding for the amino acid sequence of the formula [I] or an amino acid sequence of a partially modified formula (I] or their parts.

5. ) A DNA according to claim 4» containing a nucleotide sequence of the formula [IIJ or parts thereof. atg-yzg-xcy-cau-cau-tay-cag-cag-cau-cgt-zga-aaa-ttt-gca-gctGCC-TTC-YTG-GCA-TTV-ATT-TTC-ATZ-YTG-GCA-GCT-GTG-GAY-ACT-GCTgau-gcz-ggg-aag-aaa-gag-aaa-ccw-gaa-aaa-aau-gtg-aau-aag-tctGAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGY-GTG-CCY-ACC-AGY-GGUGAC-TGT-GGU-YTG-GGC-ACZ-CGG-GAG-GGC-ACT-CGV-ACT-GGZ-GCY-GAGTGC-AAU-CAn-ACC-ATG-AAG-ACY-CAG-AGA-TGT-AAG-ATC-CCY-TGC-AACTGG-AAG-AAG-CAU-TTT-GGZ-GCX-GAG-TGC-AAU-TAC-CAG-TTC-CAG-GCYTGG-GGA-GAA-TGT-GAC-CTV-AAY-ACZ-GCC-YTG-AAG-ACC-AGA-ACT-GGZAGY-CTG-AAG-CGA-GCY-CTG-CAC-AAT-GCY-GAZ-TGY-CAG-AAU-ACT-GTCACC-ATC-TCC-AAG-CCC-TGT-GGC-AAU-CTV-ACC-AAG-CCC-AAU-CCT-CAAGCU-GAU-TCW-AAG-AAG-AAG-AAA-AAG - GAA-GGC - AAG-AAA-CAG-GAG-AAGATG-CTG-GAT-TAA [II] wherein U is A or G, Y is C or T, X is G or T. Z is A or C, V is G or C and W is T or A.

6. ) A DNA according to claim 4, coding the following nucleotide sequence [II-I] ATG-TCG-TCC-CAG-CAA-TAT-CAG-CAG-CAA-CGT-AGA-AAA-TTT-GCA-GCTGCC-TTC-CTG-GCA-TTG-ATT-TTC-ATC-TTG-GCA-GCT-GTG-GAC-ACT-GCTGAG-GCC-GGG-AAG-AAA-GAG-AAA-CCT-GAA-AAA-AAG-GTG-AAA-AAG-TCTGAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGC-CTG-CCT-ACC-AGC-GGGGAC-TGT-GGA-TTG-GGC-ACC-CGG-GAG-GGC-ACT-CGC-ACT-GGC-GCC-GAGTGC-AAA-CAG-ACC-ATG-AAG-ACT-CAG-AGA-TGT-AAG-ATC-CCT-TGC-AACTGG-AAG-AAG-CAG-TTT-GGA-GCT-GAG-TGC-AAG-TAC-CAG-TTC-CAG-GCTTGG-GGA-GAA-TGT-GAC-CTC-AAT-ACC-GCC-TTG-AKG-ACC-AGA-ACT-GGCAGC-CTG-AAG-CGA-GCT-CTG-CAC-AAT-GCT-GAC-TGT-CAG-AAA-ACT-GTCACC-ATC-TCC-AAG-CCC-TGT-GGC-AAG-CTC-ACC-AAG-CCC-AAG-CCT-CAAGCG-GAG-TCA-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAGATG-CTG-GAT-TAA

7. ) A DNA according to claim 4 coding for the following nucleotide sequence [II-II] ATG-CAG-GCT-CAA-CAG-TAC-CAG-CAG-CAG-CGT-CGA-AAA-TTT-GCA-GCT GCC-TTC-TTG-GCA-TTC-ATT-TTC-ATA-CTG-GCA-GCT-GTG-GAT-ACT-GCT GAA-GCA-GGG-AAG-AAA-GAG-AAA-CCA-GAA-AAA-AAA-GTG-AAG-AAG-TCT GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGT-GTG-CCC-ACC-AGT-GGA GAC-TGT-GGG-CTG-GGC-ACA-CGG-GAG-GGC-ACT-CGG-ACT-GGA-GCT-GAG TGC-AAG-CAA-ACC-ATG-AAG-ACC-CAG-AGA-TGT-AAG-ATC-CCC-TGC-AAC TGG-AAG-AAG-CAA-TTT-GGC-GCG-GAG-TGC-AAA-TAC-CAG-TTC-CAG-GCC TGG-GGA-GAA-TGT-GAC-CTG-AAC-ACA-GCC-CTG-AAG-ACC-AGA-ACT-GGA AGT-CTG-AAG-CGA-GCC-CTG-CAC-AAT-GCC-GAA-TGC-CAG-AAG-ACT-GTC ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAA-CTG-ACC-AAG-CCC-AAA-CCT-CAA GCA-GAA-TCT-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG ATG-CTG-GAT-TAA

8. ) A plasmid vector containing a nucleotide sequence coding: for the protein of claim 1.

9. ) A method for the production of the protein of claim 1, characterized by cultivating host cells transformed by an expression vector containing a nucleotide sequence coding for a protein of claim 1.

10. ) A medicament comprising a protein as claimed in claims 1 to 3 and a pharmaceutically acceptable carrier or diluent.

11. ) 11 )

12. ) 12)

13. ) 13)

14. ) 14)

15. ) 15) 16)

16. ) A protein according to claim 1, substantially as hereinbefore described. A DNA according to claim 4, substantially as hereinbefore described. A plasmid vector according to claim 8, substantially as hereinbefore described. A method according to claim 9, substantially as hereinbefore described. A protein according to claim 1, whenever produced by a method claimed in claim 9 or 14. A medicament according to claim 10, substantially as hereinbefore described.