Pharmaceutical composition having an endoproteolytic activity; a

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide

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435226, 4352402, A61K 3848, C12P 2106, C12N 964

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active

059898569

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BRIEF SUMMARY
The invention relates to both a pharmaceutical composition having an endoproteolytic activity and a process for the (micro)biological production of a protein and for the in vitro cleavage of a protein, in particular a precursor protein by processing the protein with an endoproteolytically active enzyme.
The invention described herein is the result of a further study into the possible physiological significance of furin, a human protein described in European patent application EP-A-0 246 709, which is the expression product of the fur gene located in the genome upstream of the human fes/fps proto-oncogene. The patent application referred to and other publications by the same research group (Roebroek et al., Molec.Biol.Rep. 11, 1986, 117-125; Roebroek et al., EMBO J. 5, 1986, 2197-2202; and Schalken et al., J.Clin. Invest. 80, 1987, 1545-1549) show that on the basis of the limited DNA data then available, it was impossible to determine the function of the product of the fur gene. What could be determined was that the furin is probably a membrane-associated protein which has a function in which certain recognition structures play a role. It was also observed at the time that the fur gene is expressed as a 4.5 kb mRNA in liver, kidney, spleen, thymus and brain, whereas the expression in lung tissue is very slight; in non-small-cell lung carcinomas, on the other hand, a highly increased expression was found to occur, on the ground of which the fur gene was suggested to have a utility as a tumor marker.
Within the framework of the above research, the complete nucleotide sequence of a genomic DNA fragment of about 21 kbp containing the fur gene has meanwhile been determined (Van den Ouweland et al., Nucl.Acids Res. 17, 1989, 7101-7102), while the nucleotide sequence of the corresponding fur cDNA has also been determined (Van den Ouweland et al., Nucl. Acids Res. 18, 1990, 664). On the basis thereof it is not possible for the fur gene to be completely characterized, at both the level of genomic organization structure and the level of the encoding sequences. From these encoding nucleotides sequences, the amino acid sequence of the furin can also be derived.
A computer analysis of this amino acid sequence has now surprisingly revealed that furin is highly similar to subtilisin-like proteases as encoded in yeast by the KEX1 gene of Kluyveromyces lactis and the KEX2 gene of Saccharomyces cerevisiae, and that furin is evidently the higher-eukaryotic form (found in Man and in animals, such as monkey, cat, rat, mouse, chicken and Drosophila) of these endoproteases. More specifically it has been found that the furin exhibits a certain degree of homology with the catalytic domain of the hitherto described bacterial subtilisins (about 20 enzymes), such as thermitase of Thermoactinomyces vulgaris and subtilisin BPN' of Bacillus amyloliquefaciens, and exhibits a striking high homology with subtilisin-like proteases, such as the expression product of the KEX1 gene of the yeast Kluyveromyces lactis and the expression product of the KEX2 gene of the yeast Saccharomyces cerevisiae. The furin Seq ID NO:2), which contains 794 amino acids, exhibits in the domain of the amino acids 97 to 577 an overall homology of about 80.0% with the amino acids 123-584 of the expression product of said KEX1 gene (i.e., 41.6% identical amino acids and 38.3% conservative substitutes) and an overall homology of about 78.9% with the amino acids 134-597 of the expression product of said KEX2 gene (i.e., 39.4% identical amino acids and 39.5% conservative substitutes). These amino acid regions of the yeast proteases comprise the subtilisin-like catalytic domains. The subtilisin-like domain of furin is situated in an amino-terminal furin fragment comprising the amino acids 108-464 (Seq ID NO:3).
With regard to the subtilisin-like proteases, reference is made to the following publications: Tanguy-Rougeau et al., FEBS Letters 234, 1988, 464-470; Mizuno et al., Biochem. Biophys. Res Commun. 156, 1988, 246-254; Meloun et al. FEBS Letters 183, 1985, 195-200; Ma

REFERENCES:
Fuller et al Science 246 p. 482 Oct. 27, 1989.
Thim et al Proc Nat Acad Sci USA 83 p. 6766 1986.
Wise et al Proc Natl Acad Sci USA 87 p. 9378 1990.
Mizuno et al Biochem Biophys Res Comm 156 p. 246 1988.
Roebroek et al EMBO 5 p. 2197 1987.
Van den Ouweland et al NAR 17 p. 7101 1989.
Misumi et al NAR 18 p. 6719 1990.
Lehninger, Biochemistry, Worth Publishing, 1975.
Boehringer Mannheim Biochemical Cataloge p. 496.

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