Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-06-09
2003-08-12
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S252300, C435S320100, C435S325000, C435S410000, C536S023200
Reexamination Certificate
active
06605456
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is directed to purified and isolated novel I kappa B kinase-related kinases 1 and 2 (IKR-1 and IKR-2) polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and uses thereof.
2. Description of Related Art
The transcription factor NF-&kgr;B (nuclear factor &kgr;B) is composed of homo- or heterodimers of proteins from the Rel family of transcription factors. The major genes regulated by NF-&kgr;B are immune, viral, and inflammatory response genes (C. H. Regnier et al.,
Cell, Vol.
90: 373-383, July 1997). When NF-&kgr;B is not involved in active transcription of these genes, it is located in the cytoplasm bound to the inhibitory protein I&kgr;B which regulates the activity of NF-&kgr;B (J. A. DiDonato et al.,
Nature,
Vol. 388: 548-554, August 1997). I&kgr;B, when bound to NF-&kgr;B molecules, masks the nuclear localization signal of NF-&kgr;B, thus inactivating the protein.
In response to extracellular cytokines or other pro-inflammatory stimuli, I&kgr;B molecules are quickly phosphorylated by intracellular kinases such as I Kappa B kinases &agr; and &bgr; (IKK&agr; and IKK&bgr;). Once activated, IKK&agr; and IKK&bgr; specifically phosphorylate I&kgr;B at specific serine residues in the N-terminus or I&kgr;B alpha at serine residues 32 and 36, thereby targeting this molecule for proteolytic destruction. The outcome of I&kgr;B phosphorylation and destruction is the release and subsequent translocation of NF-&kgr;B from the cytosol to the nucleus where it engages transcriptional regulatory sites on a number of immune related and pro-inflammatory genes.
The eukaryotic protein kinases make up a large and rapidly expanding family of proteins related on the basis of homologous catalytic domains. Spurred by the development of gene cloning and sequencing methodologies, distinct protein kinase genes have been identified from a wide selection of invertebrates and lower eukaryotes, including
Drosophila, Caenorhabditis elegans, Aplysia, Hydra, Dictyostelium,
and budding (
Saccharomyces cerevisiae
) and fission (
Schizosaccharomyces pombe
) yeast. Homologous genes have also been identified in higher plants. Protein kinases, however, are not limited to the cukaryotes. Enzyme activities have been well documented in prokaryotes, but the prokaryotic protein kinase genes are not obviously homologous to those of the eukaryotes.
Given the important function of kinases in general and IKK's specifically, there is a need in the art for additional members of the kinase family. In addition, in view of the continuing interest in protein research, the discovery, identification, and roles of new proteins, such as protein kinases, are at the forefront of modern molecular biology and biochemistry. Despite the growing body of knowledge, there is still a need in the art for the identity and function of proteins having kinase activities. In addition, because there is an unmet need for therapeutic compounds which interfere with activation of NF-&kgr;B and because protein kinases are useful biochemical reagents, there is also need in the art for the continued discovery of unique members of the IKB protein kinase family and potential therapeutic targets thereof.
In another aspect, the identification of the primary structure, or sequence, of an unknown protein is the culmination of an arduous process of experimentation. In order to identify an unknown protein, the investigator can rely upon a comparison of the unknown protein to known peptides using a variety of techniques known to those skilled in the art. For instance, proteins are routinely analyzed using techniques such as electrophoresis, sedimentation, chromatography, sequencing and mass spectrometry.
In particular, comparison of an unknown protein to polypeptides of known molecular weight allows a determination of the apparent molecular weight of the unknown protein (T. D. Brock and M. T. Madigan,
Biology of Microorganisms
76-77 (Prentice Hall, 6d ed. 1991)). Protein molecular weight standards are commercially available to assist in the estimation of molecular weights of unknown protein (New England Biolabs Inc. Catalog:130-131, 1995; J. L. Hartley, U.S. Pat. No. 5,449,758). However, the molecular weight standards may not correspond closely enough in size to the unknown protein to allow an accurate estimation of apparent molecular weight. The difficulty in estimation of molecular weight is compounded in the case of proteins that are subjected to fragmentation by chemical or enzymatic means, modified by post-translational modification or processing, and/or associated with other proteins in non-covalent complexes.
In addition, the unique nature of the composition of a protein with regard to its specific amino acid constituents results in unique positioning of cleavage sites within the protein. Specific fragmentation of a protein by chemical or enzymatic cleavage results in a unique “peptide fingerprint” (D. W. Cleveland et al.,
J. Biol. Chem.
252:1102-1106, 1977; M. Brown et al.,
J. Gen. Virol.
50:309-316, 1980). Consequently, cleavage at specific sites results in reproducible fragmentation of a given protein into peptides of precise molecular weights. Furthermore, these peptides possess unique charge characteristics that determine the isoelectric pH of the peptide. These unique characteristics can be exploited using a variety of electrophoretic and other techniques (T. D. Brock and M. T. Madigan,
Biology of Microorganisms
76-77 (Prentice Hall, 6d ed. 1991)).
Fragmentation of proteins is further employed for amino acid composition analysis and protein sequencing (P. Matsudiara,
J. Biol. Chem.
262:10035-10038, 1987; C. Eckerskorn et al.,
Electrophoresis
1988, 9:830-838, 1988), particularly the production of fragments from proteins with a “blocked” N-terminus. In addition, fragmented proteins can be used for immunization, for affinity selection (R. A. Brown, U.S. Pat. No. 5,151,412), for determination of modification sites (e.g. phosphorylation), for generation of active biological compounds (T. D. Brock and M. T. Madigan,
Biology of Microorganisms
300-301 (Prentice Hall, 6d ed. 1991)), and for differentiation of homologous proteins (M. Brown et al.,
J. Gen. Virol.
50:309-316, 1980).
In addition, when a peptide fingerprint of an unknown protein is obtained, it can be compared to a database of known proteins to assist in the identification of the unknown protein using mass spectrometry (W. J. Henzel et al.,
Proc. Natl. Acad. Sci. USA
90:5011-5015, 1993; D. Fenyo et al.,
Electrophoresis
19:998-1005, 1998). A variety of computer software programs to facilitate these comparisons are accessible via the Internet, such as Protein Prospector (prospector.uscf.edu), MultiIdent (expasy.ch/sprot/multiident.html), PeptideSearch (mann.embl-heiedelberg.de/deSearch/FR_PeptideSearchForm.html), and ProFound (chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance. The programs compare these molecular weights to protein molecular weight information stored in databases to assist in determining the identity of the unknown protein. Accurate information concerning the number of fragmented peptides and the precise molecular weight of those peptides is required for accurate identification. Therefore, increasing the accuracy in determining the number of fragmented peptides and their molecular weight should result in enhanced likelihood of success in the identification of unknown proteins.
In addition, peptide digests of unknown proteins can be sequenced using tandem mass spectrometry (MS/MS) and the resulting sequence searched against databases (J. K. Eng, et al.,
J. Am. Soc. Mass Spec.
5:976-989 (1994); M. Mann and M. Wilm,
Anal. Chem.
66:4390-4399 (1994
Bird Timothy A.
Virca G. Duke
Achutamurthy Ponnathapu
Immunex Corporation
Kirschner Michael K.
Pak Yong
Sprunger Suzanne A.
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