Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a novel protein having an ability to form osteocyte differentiation and brain cell differentiation, DNA encoding the same, and a method for producing the protein by a genetic engineering method.
• Kad 0 matsu et al. (Kadomatsueta 1.1988, Biom. Biolys. Res.Comniun, Vo.151, Pages 1312-1318) are specific during the early development of mouse embryonic teratocarcinoma cells.
• TGF Transforming growth factor (activator)-indicates that it is a hormone involved in cell growth and differentiation, such as a protein belonging to ⁇ -f ⁇ -milli.
• BMP Bone Morphogenic Protein
• Osteoporosis and dementia as senile diseases have recently become a serious social problem in industrialized countries, and the number of patients is also increasing.
• genes and their products that are specifically expressed in osteogenic and brain cells have the potential to open new avenues for their treatment.
• the present inventors isolated a cDNA clone specifically expressed in a cell line MC3T3E1 having a property similar to that of a mouse calvaria-derived osteogenic cell (osteoplast) and isolated the same. We have found that DNA clones code for new proteins. The present inventors further used the cDNA encoding the novel protein derived from this mouse as a probe, and obtained the human-derived protein from a cDNA library prepared from human cells. CDNA clones to be cloned were also identified.
• the protein obtained by the present invention is a protein consisting of 168 amino acids belonging to a family of a kind of cell differentiation or growth factor called MK family.
• the mRNA encoding this protein is not expressed in mouse kidney cells, spleen cells, thymocytes, liver cells, lung cells, and skeletal muscle cells, but is strong in MC3T3E1 cells and in brain cells. It is weakly expressed in NIH3T3 cells.
• the present invention first provides an amino acid residue represented by the following sequence [I]: It consists of a protein having a group.
• a * 1 is S er force
• G 1 n
• a * 2 is S er or A 1 a
• a * 3 is Le eu or P he ⁇
• a * 4 is Asp or G lu.
• a preferred example of the protein of the present invention is a protein named mouse OSF-1 or human 0SF-1 represented by the following sequence [I-I] or [I- ⁇ ]. [ ⁇ -I]
• the present invention further comprises a DNA encoding the above sequence [I].
• a part of the amino acid residue in the sequence [I] can be modified by deleting, replacing or adding the amino acid without significantly changing the properties of the protein. Further, not only DNA encoding the entire sequence [I] or the modified sequence [I] but also DNA encoding the portion thereof are included in the scope of the present invention.
• the present invention also relates to an expression vector, which comprises a DNA encoding the above-mentioned protein and an appropriate gene promoter linked to its upstream end in the correct direction, for example, gene expression in animal cells. It is characterized by incorporating the early promoter of the T antigen of SV4G virus, which is commonly used for, and having DNA fragments necessary for the expression of other exogenous genes.
• the present invention relates to a method for producing the protein represented by the above-mentioned sequence [I], which comprises transforming a host cell with the expression vector of the present invention and culturing the transformant. It is characterized by producing the protein.
• the present invention relates to a protein obtained by culturing the transformant.
• protein refers to a protein having the entire amino acid sequence of the above [I], a protein having the entire amino acid sequence partially modified by deletion, substitution, or insertion; It consists of an array part.
• Example of DNA encoding 168 amino acid residues according to the present invention In example in Chinese hamster ovary cells (CHO) animal cells, such as, for example, by expressing linked downstream of the initial promoter of T antigen S V40 virus are properly even all 1 68 ⁇ Mi Amino Acids that It is possible to secrete and produce a protein having a partial sequence outside the host cell. In addition, since this protein does not have a site for modifying a sugar chain, it can be produced using a microorganism such as yeast or Escherichia coli. The protein thus produced, like other proteins belonging to the MK family, has the effect of promoting the growth and differentiation of osteoblasts and brain nerve cells.
• CHO Chinese hamster ovary cells
• the cDNA is used as a probe to isolate human alleles from the cDNA library of human osteoblasts and brain nerve cells, or cell lines established from these tumor lines. It is also possible.
• the protein according to the present invention is a protein specifically and strongly expressed in the cDNA library of MC3T3E1 cells, which are osteoblast-like cells established from mouse calvarial cells, Was selected in comparison to the sexual fibroblast cell line, NIH3T3.
• cDNA encoding human OSF-1 was also selected from the cDNA library of humans using the cDNA encoding mouse SF-1 obtained as a probe.
• the nucleotide sequence of those cDNAs is represented by [ ⁇ ].
• mouse brain, muscle, kidney, liver, spleen, thymus was confirmed by Northern hybridization, expression was observed only in brain cells except for MC3T3E1 cells.
• 0 S F-1 is a novel protein having the following properties.
• MK-1 MGF REGT-CGAQT Q VHC VPCN WK EFGADC
• OSF-1 YQFQAWGECD LNTA1KT TG SLKRA1HNAD CQKTVT S P CGK1T P PQ
• MK-1 Y FESWGACD GSTGTKARQG TL KARYTAQ CQET RVTKP CTSKTKS T
• OSF-1 AESKKKK EG KKQEKMLD
• MK-1 is known to be a secreted protein (or polypeptide), and has been suggested to be a differentiation factor for empironic carcinoma cells under the control of retinoic acid (Mineko Tomomura, Kenji Kadomatsu, Takashi Muramatsu, Biochemistry, 1989, Voi.61, ⁇ 9, Page 1040), and OSF-1 are very similar in structure to MK-1 except that their tissue specificities are osteoblasts and brain cells.
• the protein of the present invention may be a protein containing all of the amino acid residues of OSF-11 or a partially substituted amino acid residue of OSF-1 having substantially the same activity as OSF-1. Including.
• the protein of the present invention can be secreted as a protein consisting of the sequence [I]. Judging from the similarity with MK-1, it can be secreted as a protein with a molecular weight of around 90 after being cleaved from Gin (Q) at position ⁇ to Arg (R) at position 84. There is. Therefore, the protein of the present invention includes a protein consisting of the part of the sequence [I] and a partial amino acid residue having substantially the same activity as the protein.
• the DNA encoding the protein according to the present invention has the sequence
• a typical sequence of DNA according to the present invention is the following sequence [ ⁇ ].
• U is A or G
• Y is C or T
• X is G or T
• Z is A force
• C and V are G or C
• W is T or A.
• DNA encoding mouse OSF-1 and human OSF-1 are represented by the following sequences [ ⁇ -I] and [ ⁇ - ⁇ ].
• DNA encoding the protein according to the present invention includes those having the nucleotide sequence of the above-mentioned sequence [ ⁇ ] and degenerate isomers thereof.
• degenerate isomer shall mean DNA which has the same nucleotide sequence and encodes the same protein except that codons having a degenerate relationship are used.
• one means for obtaining the DNA is to convert nucleic acid. It is to produce according to the method of chemical synthesis.
• the DNA encoding mouse OSF-1 according to the present invention can be obtained from a mouse calvarial osteoblast MC3T3E1 (Kodama et al., 1981, Jpn. J. Oia 1 Biol., Vo.23, (Pages 899-901) Cells were seeded in 2.2 ⁇ 10 5 cells / plate in 40 Petri dishes with em diameter and the medium was changed appropriately at 9 ° C for about 9 days.After culturing, the cells became 8 ⁇ 1G 7 cells.
• Maniatis et al. Maniatis et al., 1982, Molecular Cloning: A Laoratory Manual, Cold Spring Harbor Laboratory
• After preparing total RNA purify mRNA, synthesize cDNA, clone It can also be manufactured by conversion.
• the cDNA derived from this mouse can also be produced as a probe from the cDNA library of another animal.
• the OSF-1 protein can be produced using Escherichia coli, yeast, or animal cells as the host, using the corresponding promoter. As a specific method, the method described in Maniatis et al. (Supra) can be used.
• Restriction enzymes were obtained from New England Biolabs, USA, and Takara Shuzo, and were used according to the manufacturer's instructions unless otherwise noted.
• Exit Delane kit for sequence cloning vector 3-ter PUC 118, T4 DNA ligase and bacterial phosphatase were obtained from Takara Shuzo and used according to the manufacturer's instructions.
• c DNA synthesis system ⁇ Plus was obtained from Amersham, UK, and used according to the manufacturer's instructions.
• AMV reverse transcriptase, mRNA purification kit, and Ec QRI-Not I adapter were obtained from Pharmacia, Sweden, and used according to the manufacturer's instructions. .
• Random primed DNA labeling kits were obtained from Behringer Mannheim Yamanouchi and used according to the manufacturer's instructions.
• i gtio cloning vector and invitrono zero packaging kit "Gigapack Gold” were obtained from Stratagene, USA, (Stratagene) and the manufacturer's instructions. Used according to. Transformation of Escherichia coli (E. coli) and ligation of DNA were performed according to the method described by Mania tis et al.
• FIG. 1 shows the structure of the plasmid pMC031.
• the white band indicates the cDNA insertion site of mouse OSF-1.
• the arrow indicates the ampicillin resistance gene.
• the black circles indicate the starting point of plasmid replication.
• FIG. 2 shows the structure of the plasmid PMCG31 and the cDNA inserts of the 10 deletion mutants, and the region in which the base sequence was determined.
• the solid line indicates the cDNA insert contained in each plasmid. Arrows indicate the region and direction in which the base sequence was determined.
• FIG. 3 shows the entire nucleotide sequence of the cDNA insertion site of pMCl and the amino acid sequence of mouse 0 S F-1 deduced from the nucleotide sequence.
• the underline indicates the portion derived from the EcoRI-NotI adapter. *** indicates a transcription termination codon.
• FIG. 4 shows examination of tissue-specific expression of the 0SF-1 gene by Northern plotting.
• Each lane is as follows. 1; thymus, 2; spleen, 3; brain, 4; kidney, 5; liver, 6; lung, 7; skeletal muscle, 8;
• FIG. 5 shows the structure of plasmid PHSG757.
• the white band indicates the poly A signal
• the white band with the arrow indicates the SV40T antigen early promoter
• the arrow indicates the chloramphenicol resistance gene.
• the black circle indicates the origin of plasmid replication.
• FIG. 6 shows the entire nucleotide sequence of the cDNA fragment of pHBR1 and the amino acid sequence of human OSF-1 deduced from the nucleotide sequence.
• Example 1 Mouse calvaria-derived osteoblast-like cell line
• RNA was prepared from 1.5-cells (Kodama et al., 1981, Jpn. J. Oral Biol., 23, 899-901) by the guanidine method (Mani at is, et al., Supra), and then the total RNA was prepared.
• mRNA was prepared using an mRNA purification kit. Using the prepared mRNA as type I, a cDNA synthesis system plus (Amersham)
• Double-stranded cDNA was synthesized. First, Ec0RI-N01I adapter-1 (Pharmacia) was ligated to the synthesized double-stranded cDNA using T4 DNA ligase, and then digested with EcoRI; Cloning vectors (Stratagene) were ligated using T4 DNA ligase.
• the i gtlO c DNA was linked to a, b down bi-DOO be sampled La evening Gee down Russia Nono 0 Tsu cage packaging kit "Gigapa' Kugoru de (G igapac It Gold)" using a stomach down bi collected by filtration ( The cells were packaged in a lambda phage in vitro and stored in SM buffer (Mani at is, et al., supra). This was transformed into E. coli C600 strain (National Institute of Preventive Health Gene Bank).
• a cDNA library was generated at a frequency of approximately 1.4 ⁇ 10 7 primary phage plaques per gram of cDNA.
• Example 2 Difference library of cDNA library derived from MC3T3E1 cells with NIH3T3 cells (ATCC CRL 1658). ) To select clones specific to MC3T3E1 cells and to clone the plasmid vector pUCIU. MC 3 T 3 E 1 cells own mRNA and MC 3 T 3 E
• NIH 3T3 cells mouse fibroblast cell line NIH 3T3 cells (ATCC CRL 1658) prepared in the same manner as 1 cells as type III, the cells were treated with AMV Linokin Strand Scryptase (Pharmacia), respectively.
• a radioactive cDNA probe was synthesized.
• cDNA library derived from MC3T3E1 cells about 1.5 ⁇ 10 4 cells were subjected to the plaque-hybridization method (Maniatis, et al.) Using each radioactive cDNA probe. According to the above, screening was performed sequentially to obtain 78 clones specifically hybridizing to a radioactive cDNA probe derived from MC3T3E1 cells.
• each phage DNA was purified from the lysate of these phage clones by phenol treatment followed by ethanol precipitation (Maniat is et al .; supra), digested with EcoRI, separated by agarose gel electrophoresis, c Only the DNA insert was purified (Maniatis et al., supra).
• radioactive probes derived from each clone were prepared using a random primed DNA labeling kit (Berlingermanheim Yamanouchi).
• mRNA 0.3 ⁇ of MC3T3E1 cells and NIH3T3 cells was separated by formaldehyde denaturing agarose gel electrophoresis (Mani at is, et al., Supra), and then separated by a nylon membrane (BYODYNE , Pall Bio Support, USA) (aniatis ⁇ , supra) 0
• cDNA insert of this Phage clone MC031 was digested with the restriction enzyme EcoRI, and the 5'-terminal phosphoric acid was removed by treatment with bacterial alkaline phosphatase (Takara Shuzo). It was ligated with PUC118 (Takara Shuzo) using T4 DNA ligase to obtain plasmid pMCl.
• Fig. 1 shows the structure.
• pMC031-1 After digesting pMC031 with the restriction enzymes SphI and BamHI, ten deletion mitants were prepared using a kilosequence deletion kit (Takara Shuzo). These are referred to as pMC031-1, pMC031-2, pMC031-3,..., PMC0310 (FIG. 2). Applied Biosystems, Inc., USA, (Applied Biosystems), using an automatic sequence analyzer (Model 370A), pMC031, and their displacement mutators. Approximately 300 base pairs were determined from the Hind site side of the cDNA insertion site. FIG. 3 shows the entire nucleotide sequence of the resulting cDNA insert and the amino acid sequence deduced therefrom. The protein presumed to be coded by this gene is called mouse OSF-1.
• Example 4 Examination of Tissue-Specific Expression of Mouse OSF-1 Tissue-specific expression of mouse 0SF-1 gene was examined by Northern blotting.
• the cDNA insert of pMCl was digested with EcoRI and purified by agarose gel electrophoresis (Maniatis et al., Supra), and this was used as a substrate for randomization in the Behringermannheim Yamanouchi. Radioactive probes were prepared using the primed DNA labeling kit.
• FIG. 6 shows the entire nucleotide sequence of the resulting cDNA insert and the amino acid sequence deduced therefrom.
• SF-1 was produced using the method described in JP-A-63-7288 for efficiently producing a protein in animal cells using the amplified gene. ⁇ ⁇
• cDNA encoding the same protein must be transferred to cassette vector p HSG (National Institute of Health).
• Insertion number VEH 7 was inserted between the SV40 virus T antigen early promoter and the polymerase II signal as follows. The EcoRI site upstream of the SV40T antigen early promoter of pHSG747 was eliminated by digestion with EcoRI, blunting of the terminal with the Klenow fragment (Takara Shuzo), and subsequent recombination with ligase. Was.
• the single Pst1 site between the SV40T antigen early promoter and the polyA signal sequence was replaced with a synthetic EcoRI linker (oligonucleotide) (Takara Shuzo), and a single EcoR at the same position. It has an I site.
• a plasmid pHSG757 was constructed for amplification of the in vitro gene (Fig. 5).
• An OSF-1 cDNA fragment sandwiched between the EcoRI sites was inserted in the EcoRI site in the forward direction to obtain plasmid pOFl.
• BstXI By digesting this plasmid with the restriction enzyme BstXI, an expression unit DNA fragment of 0SF-1 having asymmetric sticky ends at both ends as described below can be obtained.
• plasmid pHSGII National Institute of Health and Prevention Genebank registration number VE9
• the fragment of [m] obtained in this manner was prepared by digesting the DNA of Cosmid Vector-p HSG 293 (National Institute of Preventive Health, Gene Bank registration number VE 046) with BstXI digestion as follows: And 20: 1, and ligated to tandem by T4 ligase according to the method described in the above-mentioned JP-A-sho.
• OSF-1 cDNA fragment was used as a probe by the northern blotting method (Maniais et al., Supra). A strain that expressed the same gene with high efficiency was obtained.
• the selected OSF-1 producing strain contains 10% fetal bovine serum-MEM medium (GIBC 0) And OSF-1 was detected in the culture supernatant.
• novel protein having osteocyte differentiation-forming ability, DNA encoding the same, and a method for producing the protein by a genetic engineering method.
• the novel protein provided by the present invention has the potential to be used for the treatment of osteoporosis and dementia as senile diseases.

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• 1991
  • 1991-06-27 AU AU80784/91A patent/AU8078491A/en not_active Abandoned
  • 1991-06-27 PT PT98109A patent/PT98109A/pt not_active Application Discontinuation
  • 1991-06-27 IL IL98653A patent/IL98653A0/xx unknown
  • 1991-06-27 WO PCT/JP1991/000871 patent/WO1992000324A1/ja unknown
  • 1991-06-28 IE IE228191A patent/IE912281A1/en unknown

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