Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1999-06-04
2001-05-29
Nashed, Nashaat T. (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C435S219000, C536S023500
Reexamination Certificate
active
06239254
ABSTRACT:
BACKGROUND OF THE INVENTION
Protein inhibitors are classified into a series of families based on extensive sequence homologies among the family members and the conservation of intrachain disulfide bridges, see Laskowski and Kato,
Ann. Rev. Biochem
. 49: 593-626 (1980). An example of a serine proteinase inhibitor is the serine proteinase inhibitor aprotinin which is used therapeutically in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction. Administration of aprotinin in high doses significantly reduces blood loss in connection with cardiac surgery, including cardiopulmonary bypass operations.
However, when administered in vivo, aprotinin has been found to have a nephrotoxic effect in rats, rabbits and dogs after repeated injections of relatively high doses. The nephrotoxicity (appearing, i.e., in the form of lesions) observed for aprotinin might be ascribed to the accumulation of aprotinin in the proximal tubulus cells of the kidneys as a result of the high positive net charge of aprotinin, which causes it to be bound to the negatively charged surfaces of the tubuli. This nephrotoxicity makes aprotinin less suitable for clinical purposes, particularly in those uses requiring administration of large doses of the inhibitor (such as cardiopulmonary bypass operations). Furthermore, aprotinin is a bovine protein, which may induce an immune response upon administration to humans.
Thus there is a need for serine proteinase inhibitors which are not toxic for the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction.
SUMMARY OF THE INVENTION
The present invention fills this need by providing for a new class of proteinase inhibitors called disulfide core proteinase inhibitors (hereinafter referred to as a Zdsc1 polypeptide). Murine Zdsc1, SEQ ID NOs: 1 and 2 has a signal sequence extending from the methionine at position 1 through and including the alanine at position 24 of SEQ ID NO:2. The mature murine Zdsc1 polypeptide is also depicted by SEQ ID NO:3. SEQ ID NO:4 and 5 are examples of a mature human Zdsc1 polypeptide and polynucleotide which encodes it. A generic Zdsc1 polypeptide is exemplified by SEQ ID NO:6.
Within one aspect of the invention there is provided an isolated polypeptide. The polypeptide being comprised of a sequence of amino acids containing the sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:5.
Within another aspect of the invention there is provided an isolated polynucleotide which encodes a polypeptide comprised of a sequence of amino acids containing the sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:5.
Within an additional aspect of the invention there is provided a polynucleotide sequence which hybridizes under stringent conditions to either SEQ ID NO:1 or SEQ ID NO:4 or to a complementary sequence of SEQ ID NO:1 or to a complementary sequence of SEQ ID NO:4.
Within an additional aspect of the invention there is provided a polynucleotide sequence which is at least 90%, 95%, or 99% homologous to a polynucleotide sequence which encodes the polypeptide of SEQ ID NO:3 or SEQ ID NO:4.
Within another aspect of the invention there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a Zdsc1 polypeptide, containing an amino acid sequence as described above.
Within another aspect of the invention there is provided a cultured eukaryotic, bacterial, fungal or other cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a mammalian Zdsc1 polypeptide encoded by the DNA segment.
Within another aspect of the invention there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond. The first portion of the chimeric polypeptide consists essentially of a Zdsc1 polypeptide as described above. The invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
Within an additional aspect of the invention there is provided an antibody that specifically binds to a polypeptide as disclosed above and an anti-idiotypic antibody of an antibody which specifically binds to a Zdsc1 antibody.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
All references cited herein are incorporated in their entirety herein by reference.
Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms.
The term “affinity tag” is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A, Nilsson et al.,
EMBO J
. 4:1075 (1985); Nilsson et al.,
Methods Enzymol
. 198:3 (1991), glutathione S transferase, Smith and Johnson,
Gene
67:31, 1988), Glu-Glu affinity tag, Grussenmeyer et al.,
Proc. Natl. Acad. Sci. USA
82:7952-4 (1985), substance P, Flag™ peptide (Hopp et al.,
Biotechnology
6:1204-10 (1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al,
Protein Expression and Purification
2: 95-107 (1991). DNAs encoding affinity tags are available from commercial suppliers, (e.g., Pharmacia Biotech, Piscataway, N.J.).
The term “allelic variant” is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
The terms “amino-terminal” and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
The term “complement/anti-complement pair” denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <10
9
M
−1
.
The term “complements of a polynucleotide molecule” is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.
The term “contig” denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to “overlap” a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide.
The term “degenerate nucleotide sequence” denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a refe
Fronda Christian L.
Lunn, Esq. Paul G.
Nashed Nashaat T.
ZymoGenetics Inc.
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