Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-06-17
2003-07-01
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C424S094100, C424S094640, C435S183000, C530S350000, C530S381000, C930S010000
Reexamination Certificate
active
06586390
ABSTRACT:
1. TECHNICAL FIELD
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods. In particular, the invention relates to a novel human prothrombinase-like polypeptide.
2. BACKGROUND ART
Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.
Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences. It is to these polypeptides and the polynucleotides encoding them that the present invention is directed. In particular, this invention is directed to a novel prothrombinase-like polypeptides and polynucleotides.
3. SUMMARY OF THE INVENTION
The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies. Specifically, the polynucleotides of the present invention are based on polynucleotide isolated from cDNA library prepared from human ovary (Hyseq clone identification number 7735656).
The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in the SEQ ID NO: 1-3; a polynucleotide comprising any of the full length protein coding sequences of the SEQ ID NO: 1-3; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of the SEQ ID NO: 1-3. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in the SEQ ID NO: 1-3; (b) a nucleotide sequence encoding SEQ ID NO: 4-9; a polynucleotide which is an allelic variant of any polynucleotides recited above; a polynucleotide which encodes a species homolog (e.g. orthologs) of any of the proteins recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising SEQ ID NO: 4-9.
The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-3. The sequence information can be a segment of any one of SEQ ID NO: 1-3 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-3. One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one set of chromosomes. Because 4
°
possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosome. Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5. When these segments are used in arrays for expression studies, fifteen-mer segment can be used. The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence. Preferably, the nucleic acid fragment or subsequence comprise SEQ ID NO: 1.
Similarly, when using a sequence information for detecting a single mismatch, a segment can be a twenty-five mer. The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷4
25
) times the increased probability for mismatch at each nucleotide position (3×25). The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five. The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.
A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or unique identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.
This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.
Human prothrombinase-like (“Prothrombinase-like”) polypeptide or (SEQ ID NO: 4) is an approximately 388-amino acid protein with a predicted molecular mass of approximately 43 kDa unglycosylated. Protein database searches with the BLAST algorithm indicate that SEQ ID NO: 4 is homologous to human prothrombinase Fg12 protein.
FIG. 1
shows the BLASTX amino acid sequence alignment between SEQ ID NO: 4 (also identified as “Prothrombinase-like”) and human prothrombinase Fg12 protein (“Prothrombinase Fg12”), indicating that the two sequences share 53% similarity over 290 amino acid residues of SEQ ID NO: 4 and 37% identity over 290 amino acid residues of SEQ ID NO: 4. The sequences of the present invention are expected to have prothrombinase activity, as defined below.
A predicted approximately twenty residue signal peptide is encoded from approximately residue 1 to residue 20 of SEQ ID NO: 4. The mature portion (also represented as SEQ ID NO: 9) is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product. The signal peptide was predicted using the Kyte/Doolittle algorithm (J. Molecular Biology, vol. 157, pp. 105-131 (1982), incorporated herein by reference). One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
Using eMATRIX software package (Stanford University, Stanford, Calif.), Prothrom
Arterburn Matthew C.
Boyle Bryan J.
Drmanac Radoje T.
Haley Dana A.
Ho Alice S.
Hyseq Inc.
Low Christopher S. F.
Schnizer Holly
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