Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-08-09
2003-02-04
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C530S300000, C530S350000
Reexamination Certificate
active
06514741
ABSTRACT:
BACKGROUND OF THE INVENTION
Human tryptase is a serine protease with trypsin-like proteolytic activity. For a general review see Numerof, R. P. et al.,
Exp. Opin. Invest. Drugs
6:811-817, 1997; Chan, H. et al.,
Prot. Express. Purif
. 15:251-257, 1999; Elrod, K. C., and Numerof, R. P.,
Emerging Therapeutic Targets
3:203-212, 1999; and Clark, J. M. et al.,
Drugs of the Future
21:811-816, 1996. There are at least 2 genes for tryptase in the human genome that encode &agr;-tryptase and &bgr;-tryptase which are about 93% identical. Human tryptase has a tetrameric structure that requires heparin for stability. Each of the subunits are catalytically active.
Human tryptase is almost exclusively found in the secretory granules of mast cells and is released along with heparin and histamine upon mast cell activation in inflammatory response. Mast cells containing tryptase have been identified in lung, gut mucosa, and skin mast cells. Human tryptase has been established as an important mediator of airway response and is implicated in increasing the magnitude of broncoconstriction in asthma. Tryptase has neuropeptidase activity that may directly induce asthmatic response by degrading broncoactive neuropeptides having broncodilatory activities hence increasing broncoconstriction. Neuropeptides cleaved by tryptase include vasoactive intestinal peptide (VIP), and peptide histidine methionine (PHM). In addition, tryptase has been shown to stimulate cytokine production, and exhibit mitogenic effects in a variety of cell types including human and rat lung fibroblasts, human bronchial epithelial cells, and dog airway smooth muscle cells and hence may also contribute to the hyperplasia and fibrotic changes observed in diseases such as asthma. Moreover, tryptase inhibitors have been found effective in reducing asthmatic response to exposure of antigen in animal models (allergic sheep) and in human trials, and in blocking the mitogenic effects described above.
Human tryptase has been implicated in other activities such as cleaving fibrinogen &agr; and &bgr; chains, collagen IV, gelatinase and fibronectin. As such it is implicated in inhibition of coagulation and in tissue remodeling in the lung and other tissues. In addition, tryptase is shown to cleave calcitonin gene-related peptide (CGRP) which is a potent vasodilator and hence may potentiate gastric ulceration or increase cutaneous neurogenic inflammation, and promote smooth muscle contraction. In addition, tryptases are implicated in matrix degradation, wound healing and tumor metastasis.
There is a continuing need to discover new tryptase homologs, related serine proteases, and the like. The in vivo activity of human tryptase illustrates the enormous clinical potential of, and need for, related polypeptides, their agonists, and antagonists. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
Within one aspect, the present invention provides an isolated polynucleotide encoding a serine protease polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 44 (Val) to amino acid number 276 (Ile); (b) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 43 (Val) to amino acid number 275 (Arg); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Leu) to amino acid number 276 (Ile); (d) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 19 (Arg) to amino acid number 275 (Arg); (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 44 (Val) to amino acid number 314 (Leu); (f) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 43 (Val) to amino acid number 312 (Leu); (g) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 24 (Leu) to amino acid number 314 (Leu); (h) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 19 (Arg) to amino acid number 312 (Leu); (i) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 314 (Leu), (j) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 1 (Met) to amino acid number 312 (Leu), and (k) the amino acid sequence as shown in SEQ ID NO:24; and wherein the amino acid percent identity is determined using a FASTA program with ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62, with other parameters set as default. In one embodiment, the isolated polynucleotide disclosed above encodes a serine protease polypeptide that comprises a sequence of amino acid residues an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 44 (Val) to amino acid number 276 (Ile); (b) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 43 (Val) to amino acid number 275 (Arg);.(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Leu) to amino acid number 276 (Ile); (d) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 19 (Arg) to amino acid number 275 (Arg); (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 44 (Val) to amino acid number 314 (Leu); (f) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 43 (Val) to amino acid number 312 (Leu); (g) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 24 (Leu) to amino acid number 314 (Leu); (h) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 19 (Arg) to amino acid number 312 (Leu); (i) the amino acid sequence as shown in SEQ ED NO:2 from amino acid number 1 (Met) to amino acid number 314 (Leu), (j) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 1 (Met) to amino acid number 312 (Leu), and (k) the amino acid sequence as shown in SEQ ID NO:24. In another embodiment, the isolated polynucleotide disclosed above encodes a serine protease polypeptide that has protease activity. In another embodiment, the isolated polynucleotide disclosed above encodes a serine protease polypeptide consisting of a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid number 44 (Val) to amino acid number 276 (Ile) or of a sequence of amino acid residues as shown in SEQ ID NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg).
Within a second aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a serine protease polypeptide with an amino acid sequence consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 44 (Val) to amino acid number 276 (Ile); (b) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 43 (Val) to amino acid number 275 (Arg); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Leu) to amino acid number 314 (Leu); (d) the amino acid sequence as shown in SEQ ID NO:15 from amino acid number 19 (Arg) to amino acid number 312 (Leu); and (e) the amino acid sequence as shown in SEQ ID NO:24; and a transcription terminator. Within one embodiment, the expression vector disclosed above further comprises a secretory signal sequence operably linked to the DNA segment.
Within a third aspect, the present invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA segment.
Within another aspect, the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide that is selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 2 from residue number 1 (Met), to residue
Presnell Scott R.
Taft David W.
Johnson Jennifer K.
Loeb Bronwen M.
Yucel Remy
ZymoGenetics Inc.
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