Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
2000-08-29
2003-10-28
Nolan, Patrick J. (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S139100, C530S387900, C435S007100, C435S810000
Reexamination Certificate
active
06638507
ABSTRACT:
FIELD OF THE INVENTION
The present invention contemplates compositions related to proteins from animals, e.g., mammals, which function as proteases. In particular, it provides nucleic acids which encode, antibodies to, and proteins which exhibit biological functions, e.g., capacity to degrade proteinaceous substrates.
BACKGROUND OF THE INVENTION
The proteases are a very broad group of enzymes which carry out an enzymatic function of hydrolysing a peptide bond. Within the group, there is a wide range of substrate specificities for the amino acids adjacent the cleavage sites. Proteases are typically categorized on the basis of their catalytic mechanisms, e.g., based upon studies of their active sites, or by the effects of pH. Four main categories of proteases are serine proteinases, sulfhydryl proteases, acid proteases, and metalloproteases. They may also be classified according to their cleavage sites, e.g., endoproteases, amino peptidases, or carboxy peptidases.
Proteases have traditionally held a large share of the industrial enzyme market. Proteases are used in many industrial processes, including in detergents and cleaning products, e.g., to degrade protein materials such as blood and stains, in leather production, e.g., to remove hair, in baking, e.g., to break down glutens, in flavorings, e.g., soy sauce, in meat tenderizing, e.g., to break down collagen, in gelatin or food supplement production, in the textile industry, in waste treatment, and in the photographic industry. See, e.g., Gusek (1991)
Inform
1:14-18; Zamost, et al. (1996)
J. Industrial Microbiol
. 8:71-82; James and Simpson (1996)
CRC Critical Reviews in Food Science and Nutrition
36:437-463; Teichgraeber, et al. (1993)
Trends in Food Science and Technology
4:145-149; Tjwan, et al. (1993)
J. Dairy Research
60:269-286; Haard (1992)
J. Aquatic Food Product Technology
1:17-35; van Dijk (1995)
Laundry and Cleaning News
21:32-33; Nolte, et al. (1996)
J. Textile Institute
87:212-226; Chikkodi, et al. (1995)
Textile Res. J
. 65:564-569; and Shih (1993)
Poultry Science
72:1617-1620.
While there are many uses for proteases, there is always the need for a more active protease under various specific conditions. There is a need for new proteinases of differing properties, specificities, and activities.
SUMMARY OF THE INVENTION
The present invention provides a composition of matter selected from an antibody binding site which specifically binds to a human APG04, FDH02, or D1B2 protein or significant fragment thereof; an expression vector encoding a human APG04, FDH02, or D1B2 protein or significant fragment thereof; a substantially pure protein which is specifically recognized by the antibody binding site; and a substantially pure APG04, FDH02, or D1B2 protein or peptide thereof, or a fusion protein comprising at least a 30 amino acid fragment of human APG04, FDH02, or D1B2 protein sequence.
In the antibody binding site embodiments, the antibody binding site may be: specifically immunoreactive with a mature protein selected from the group consisting of the polypeptides of SEQ ID NO: 2, 4, and 6; raised against a purified or recombinantly produced human APG04, FDH02, or D1B2 protein; in a monoclonal antibody, Fab, or F(ab)2; or in a detectably labeled antibody. In certain embodiments; the antibody binding site is detected in a biological sample by a method of: contacting a binding agent having an affinity for the human APG04, FDH02, or D1B2 protein with the biological sample; incubating the binding agent with the biological sample to form a binding agent:human APG04, FDH02, or D1B2 protein complex; and detecting the complex. In a preferred embodiment, the biological sample is from a human, and the binding agent is an antibody.
A kit embodiment is provided comprising a composition, described above, with either instructional material for the use and/or disposal of the composition and products; or segregation of the composition into a compartment or container.
A nucleic acid embodiment of the invention includes an expression vector encoding a human APG04, FDH02, or D1B2 protein, wherein the protein specifically binds an antibody generated against an immunogen selected from the mature polypeptide portions of SEQ ID NO: 2, 4, and 6. The vector may: encode a human APG04, FDH02, or D1B2 polypeptide with complete sequence identity to a naturally occurring human APG04, FDH02, or D1B2 protein; encode a human APG04, FDH02, or D1B2 protein comprising sequence selected from the polypeptides of SEQ ID NO: 2, 4, and 6; or comprise sequence selected from the nucleic acids of SEQ ID NO: 1, 3, or 5. In other embodiments, the vector is capable of selectively hybridizing to a nucleic acid encoding a natural human APG04, FDH02, or D1B2 protein, e.g., a mature protein coding segment of SEQ ID NO: 1, 3, or 5. In various preferred embodiments, the isolated nucleic acid is detected in a biological sample by a method: contacting a biological sample with a nucleic acid probe capable of selectively hybridizing to the nucleic acid; incubating the nucleic acid probe with the biological sample to form a hybrid of the nucleic acid probe with complementary nucleic acid sequences present in the biological sample; and determining the extent of hybridization of the nucleic acid probe to the complementary nucleic acid sequences. In such method, preferably the nucleic acid probe is capable of hybridizing to a nucleic acid encoding a protein consisting of the polypeptides of SEQ ID NO: 2, 4, or 6.
In protein embodiments, the human APG04, FDH02, or D1B2 protein specifically binds to an antibody generated against the respective immunogen; e.g., the polypeptides of SEQ ID NO: 2, 4, or 6. In various embodiments, the isolated human APG04, FDH02, or D1B2 protein consists of a polypeptide comprising sequence from SEQ ID NO: 2, 4, or 6; is recombinantly produced, or is a naturally occurring protein.
The present invention also embraces a cell transfected with the isolated or recombinant nucleic acid encoding a human APG04, FDH02, or D1B2 protein, e.g., where the nucleic acid has SEQ ID NO: 1, 3, or 5.
DETAILED DESCRIPTION
OUTLINE
I. General
II. Definitions
III. Nucleic Acids
IV. Making APG04, FDH02, or D1B2 Protein
V. Antibodies
a. antibody production
b. immunoassays
VI. Purified APG04, FDH02, and D1B2 Protein
VII. Physical Variants
VIII. Binding Agent: APG04, FDH02, and D1B2 Protein Complexes
IX. Functional Variants
X. Uses
XI. Kits
XII. Substrate Identification
I. General
The present invention provides DNA sequences encoding mammalian proteins which exhibit structural properties or motifs characteristic of a protease. The proteases described herein are designated APG04, FDH02 and D1B2. See Tables 1, 2, and 3.
The descriptions below are directed, for exemplary purposes, to primate embodiments, e.g., human, but are likewise applicable to related embodiments from other, e.g., natural, sources. These sources should, where appropriate, include various vertebrates, typically warm blooded animals, e.g., birds and mammals, particularly domestic animals, and primates.
TABLE 1
Human APG04 nucleotide and predicted amino acid sequence. SEQ ID
NO: 1 and 2. The predicted leader sequence is underlined. The mature
peptide probably begins about at Pro (position 21). Active site/zinc
chelating residues are indicated by •.
TTGATGGCCA CCAGGTGATC TCTGGTCTCT TCAGTGTGGC TTTGCAGACT ATAAAGGCGC
60
AGCGCGCCAA CGAGGCGGGT TGGCCCCAGA CGGCGGAGAG GAAGGGCAGA GTCGGCGGTC
120
CTGAGACTTG GGGCGGCCCC TTGGAGGTCA GCCCCGCTCG CTCCTCCCGG CCCTCTCCTC
180
CTCTCCGAGG TCCGAGGCGG GCAGCGGGCT GTGGGCGGGC AGGAGGCTGC GGAGGGGCGG
240
GGGGCAGGAA GGGGCGGGGG GCTCGGCGCA CTCGGCAGGA AGAGACCGAC CCGCCACCCG
300
CCGTAGCCCG CGCGCCCCTG GCACTCAATC CCCGCC ATG TGG GGG CTC CTG CTC
354
Met Trp Gly Leu Leu Leu
&
Balasubramanian Sriram
Ford John
Gorman Daniel M.
Zurawski Gerard
Brody Tom
Ching Edwin P.
Nolan Patrick J.
Schering Corporation
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