| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 02.04.2002 |
| PATENT TITLE |
DNA coding for a human vasoconstrictive peptide and use thereof |
| PATENT ABSTRACT |
Disclosed are (1) a DNA containing a cDNA segment coding for human endothelin-2 (SEQ ID NO:1), (2) a precursor of human endothelin-2 (SEQ ID NO:2), (3) a transformant carrying a DNA containing a cDNA segment coding for human endothelin-2, and (4) a method for preparing mature human endothelin-2 which comprises culturing the transformant described in (3), accumulating a protein in a culture medium, and collecting the same, whereby human endothelin-2 and the precursors thereof can be produced in large amounts. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 10, 1991 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
M. Yanagisawa, Nature, 332:411-415 (1988). Y. Itoh, et al., FEBS Letters, 231:440-444 (1988). A. Inoue, et al., Proc. Natl. Acad. Sci. USA., 86:2863-2867 (1989). Onkubo, S. et al. "Specific expression of human endothelin-2 . . . " FEBS Lett. 274: 136-140 (Nov. 1990). Kashiwabara, T. "Putative precursors of endothelin have . . . " FEBS Lett. 247: 73-76 (Apr. 1989). Gluschankof, P. "Role of peptide substrate Structure in the . . . " FEBS Lett 234: 149-152 (Jul. 1988). Berger et al. (ed.) "Guide to Molecular Cloning Techniques" Meth. in Enzymol 152: pp. 393-399, 415-423, 433-447, 661-704 (1987). Japanese Journal of Pharmacology Supplement 1, vol. 52, Mar. 25, 1990., Kyoto JP p. 112P--M. Yanagisawa, et al.* European Journal of Pharmacology, vol. 183, No. 5, Jul. 5, 1990, Amsterdam N1 pp. 1628-1629--M. Yanagisawa, et al.* FEBS Letters, ovl. 274, No. 1-2, Nov. 12, 1990, Amsterdam NL pp. 136-140--S. Ohkubo, et al.* Genomics, vol. 10, No. 1, May, 1991, San Diego, US pp. 236-242--K.D. Bloch, et al. |
| PATENT CLAIMS |
What is claimed is: 1. An isolated and purified precursor protein having an amino acid sequence as defined in the Sequence Listing by SEQ ID NO:2. 2. An isolated and purified DNA coding for human endothelin-2 having a nucleotide sequence represented by the nucleotide sequence of Nos. 59 to 592 of SEQ ID NO:1. 3. A transformant carrying a DNA containing a cDNA segment coding for human endothelin-2 in which said cDNA segment coding for human endothelin-2 contains a nucleotide sequence as defined in the Sequence Listing by SEQ ID NO:1. 4. A transformant which has the characteristics of Escherichia coli MV1183/pHET-2(K) (FERM-BP-3008). 5. A transformant which has the characteristics of Escherichia coli MC 1061/p3/pTS612 (FERM-BP-3211). 6. A method for preparing a mature human endothelin-2 protein which comprises culturing the transformant claimed in claim 3, accumulating the mature endothelin-2 protein in a culture medium, and collecting the same. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to a DNA containing a cDNA segment coding for a human vasoconstrictive peptide (SEQ ID NO:1), namely human endothein-2, a precursor protein of human endothelin-2 (SEQ ID NO:2) and a method for preparing endothelin-2. In this specification, the term "precursor protein" is preferably used to describe a protein which includes an amino acid sequence of a mature peptide and has a portion or all of an amino acid sequence coded with a DNA segment of the peptide at the N-terminus, the C-terminus or both termini thereof. BACKGROUND OF THE INVENTION There have been reports of endothelium-dependent vasoconstrictor reactions to various mechanical and chemical stimuli as well as endothelium-dependent vasodilative reactions. For example, it is known that vasoconstriction can be induced by mechanical loads such as vascular stretch and increased vascular inner pressure, by such agents as thrombin and by hypoxemia, and further that noradrenaline-induced vasoconstriction can be enhanced by use of neuropeptide Y [Proc. Natl. Acad. Sci. U.S.A. 79, 5485 (1982); ibid. 81, 4577 (1984)]. Endothelial cell-derived coronary vascular constrictor factors (each having molecular weights of 8,500 and 3,000) are described in Amer. J. Physiol. 248, c550 (1985) and J. Cell Physiol. 132, 263 (1987). However, their sequences are unknown. An endothelial cell-derived peptide-like substance is also described in J. Pharmacl. Exp. Ther. 236, 339 (1985). However, the sequence of that substance is also unknown. On the other hand, vasopressin is known as a peptide having a vasoconstrictor activity. The amino acid sequence of vasopression has been determined. There have been no reports, however, that vasopressin was obtained from mammalian or bird vascular endothelial cells. Although there is a report that an angiotensin having a vasoconstrictor activity was obtained from the endothelial cells of bovine aortas [Circulation Research 60, 422 (1987)], the angiotensin is a peptide having a molecular weight of only about 1,000. Some of the present inventors have previously succeeded in isolating porcine endothelin as a peptide having a similar vasoconstrictor activity from the endothelial cells of porcine aortas (Japanese Patent Application No. 255381/1987). Some of the present inventors have also succeeded in isolating human endothelin and cloning porcine endothelin cDNA and human endothelin cDNA (Japanese Patent Application Nos. 275613/1987, 313155/1987 and 148158/1988). The mature polypeptides of the porcine endothelin and the human endothelin have the same amino acid sequence, and are referred to as endothelin-1. Further, the present inventors have filed patent applications with respect to the isolation of rat endothelin and the cloning of its cDNA (Japanese Patent Application Nos. 174935/1988 and 188083/1988), and this is referred to as endothelin-3. Furthermore, the present inventors have also filed a patent application with respect to the cloning of human endothelin-3 (Japanese Patent Application No. 278497/1989). Moreover, the present inventors have also filed a patent application with respect to the isolation of mouse endothelin and the cloning of its cDNA (Japanese Patent Application No. 223389/1988), and this is referred to as endothelin B. In addition, the present inventors have cloned, from a genomic human library, a DNA coding for endothelin having an amino acid sequence different from that of endothelin-1 which has been named endothelin-2, and have filed a patent application with respect to a protein of endothelin-2 and its DNA (Japanese Patent Application No. 274990/1989). The amino acid sequences of these endothelin-1 (SEQ ID NO:3), endothelin B (SEQ ID NO:4), endothelin-3 (SEQ ID NO:5) and endothelin-2 (SEQ ID NO:6) are shown in FIG. 1 in comparison to one another. Endothelin is a general term for peptides having a molecular weight of 2500.+-.300 and having 21 amino acid residues, including four cysteine groups located at the 1st, 3rd, 11th and 15th residues from the N-terminus of the amino acid sequence, which form two sets of disulfide bonds. One of the combinations of the disulfide bonds may be 1-15 and 3-11 cysteine groups, and the other may be 1-11 and 3-15. The former combination is higher in ratio of formation and in activity. than the latter combination. As described above, homologous endothelin peptides have been discovered from various animals. However, no novel homologous genes have been discovered from the same animal species. It is therefore a current subject that novel homologous endothelin is further screened, and the structure and activity of the endothelin are studied, thereby examining its usefulness, and that the novel peptide is cloned by recombinant DNA technology to allow mass production thereof. SUMMARY OF THE INVENTION The present inventors have variously studied, considering that important contributions will be made to future studies and medical treatments, if a novel homologous gene having the vasoconstrictor activity described above can be isolated and further prepared by recombinant DNA technology. As a result, the following information has been obtained, thus arriving at the present invention. Namely, the present inventors have succeeded in cloning cDNA (complementary DNA) coding for endothelin having an amino acid sequence different from those of the above endothelin-1 and endothelin-3 [human endothelin (endothelin A) and endothelin-3] from a human cDNA library by using as a probe the synthesized DNA segment coding for a portion of genomic DNA of the human endothelin described in the patent applications previously filed and a DNA comprising an about 99-bp genomic DNA segment of endothelin-2, and consequently in pioneering its mass production by recombinant technology. The present inventors further provide a precursor of human endothelin-2 having a novel amino acid sequence. In accordance with the present invention, there are provided (1) a DNA containing a cDNA segment coding for human endothelin-2, (2) a precursor of human endothelin-2, (3) a transformant carrying a DNA containing a cDNA segment coding for human endothelin-2, and (4) a method for preparing mature human endothelin-2 which comprises culturing the transformant described in (3), accumulating a protein in a culture medium, and collecting the same. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows amino acid sequences of various endothelin peptides in comparison to one another; FIG. 2 shows a restriction enzyme fragment map of a cDNA segment coding for human endothelin-2 obtained in the present invention; FIGS 3-1 to 3-3 shows a nucleotide sequence (SEQ ID NO:1) of the cDNA segment coding for human endothelin-2 obtained in the present invention and an amino acid sequence (SEQ ID NO:2) ascertained from that nucleotide sequence; FIG. 4 shows an amino acid sequence of a precursor of human endothelin-2 (SEQ ID NO:2); FIG. 5 is a schematic representation showing the construction of a plasmid for expression of human endothelin in Example 5; and FIG. 6 is a graph relating to expression of human endothelin of the present invention. FIG. 7 is a reverse phase high performance chromatogram relating to expression of human endothelin-2 of the present invention and precursor thereof. FIG. 8 is a mass spectrogram of human big endothelin-2 (1-38). |
| PATENT PHOTOCOPY | Available on request |
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