Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-06-23
2002-05-14
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C435S252300, C435S255100, C435S325000, C435S419000, C435S320100, C435S348000, C536S023200
Reexamination Certificate
active
06387676
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to purified and isolated human polypeptides having kinase function, 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, the use of such polypeptides and fragmented peptides in phosphorylation reactions and as molecular weight markers, the use of such polypeptides and fragmented peptides as controls for peptide fragmentation, the use of such polypeptides in screening assays, and kits comprising these reagents.
BACKGROUND OF THE INVENTION
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 eukaryotes. Enzyme activities have been well documented in prokaryotes, but the prokaryotic protein kinase genes are not obviously homologous to those of the eukaryotes. Because protein kinases are useful biochemical reagents, there is a need in the art for the continued discovery of unique members of the protein kinase family.
In addition, the discovery and identification of proteins are at the forefront of modern molecular biology and biochemistry. The identification of the primary structure, or sequence, of a sample protein is the culmination of an arduous process of experimentation. In order to identify an unknown sample protein, the investigator can rely upon comparison of the unknown sample 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, and mass spectrometry.
Comparison of an unknown protein sample to polypeptides of known molecular weight allows a determination of the apparent molecular weight of the unknown protein sample (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 samples (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 sample 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 (A. L. Lehninger,
Biochemistry
106-108 (Worth Books, 2d ed. 1981)). Chemical fragmentation can be achieved by incubation of a protein with a chemical, such as cyanogen bromide, which leads to cleavage of the peptide bond on the carboxyl side of methionine residues (E. Gross,
Methods in Enz
. 11:238-255, 1967). Enzymatic fragmentation of a protein can be achieved by incubation of a protein with a protease that cleaves at multiple amino acid residues (D. W. Cleveland et al.,
J. Biol. Chem
. 252:1102-1106, 1977). Enzymatic fragmentation of a protein can also be achieved by incubation of a protein with a protease, such as Achromobacter protease I (F. Sakiyama and A. Nakata, U.S. Pat. No. 5,248,599; T. Masaki et al.,
Biochim. Biophys. Acta
660:44-50, 1981; T. Masaki et al.,
Biochim. Biophys. Acta
660:51-55, 1981), which leads to cleavage of the peptide bond on the carboxyl side of lysine residues. The molecular weights of the fragmented peptides can cover a large range of molecular weights and the peptides can be numerous. Variations in the degree of fragmentation can also be accomplished (D. W. Cleveland et al.,
J. Biol. Chem
. 252:1102-1106, 1977).
The unique nature of the composition of a protein with regard to its specific amino acid constituents results in a 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)).
When a peptide fingerprint of an unknown protein is obtained, this can be compared to a database of known proteins to assist in the identification of the unknown protein (W. J. Henzel et al.,
Proc. Natl. Acad. Sci. USA
90:5011-5015, 1993; B. Thiede et al.,
Electrophoresis
1996, 17:588-599, 1996). A variety of computer software programs are accessible via the Internet to the skilled artisan for the facilitation of such comparisons, such as MultiIdent (Internet site: www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site: www.mann.emblheiedelberg.de...deSearch/FR_PeptideSearchForm.html), and ProFound (Internet site:www.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 databases to assist in the elucidation of the identity of the sample 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 the determination of the number of fragmented peptides and the precise molecular weight of those peptides should result in enhanced success in the identification of unknown proteins.
Fragmentation of proteins is further employed for the production of fragments for amino acid composition analysis and protein sequencing (P. Matsudiara,
J. Biol. Chem
. 262:10035-10038, 1987; C. Eckerskom et al.,
Electrophoresis
1988, 9:830-838, 1988), particularly the production of fragments from proteins with a “blocked” N-terminus. In addition, fragmentation of proteins can be used in the preparation of peptides for mass spectrometry (W. J. Henzel et al.,
Proc. Natl. Acad Sci. USA
90:5011-5015, 1993; B. Thiede et al.,
Electrophoresis
1996, 17:588-599, 1996), 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 ofMicroorganisms
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 view of the continuing interest in protein research and the elucidation of protein structure and properties, there exists a need in the art for polypeptides having kinase function or suitable for use in peptide fragmentation studies and in molecular weight measurements.
SUMMARY OF THE INVENTION
The invention aids in fulfilling these needs in the art. The invention encompasses an isolated human nucleic acid molecule comprising the DNA sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or 15 and an isolated human nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 14, or 16. The invention also encompasses nucleic acid molecules complementary to these sequences. As such, the invention includes double-stranded nucleic acid mo
Anderson Dirk M.
Bird Timothy A.
Marken John S.
Virca G. Duke
Immunex Corporation
Prouty Rebecca E.
Rao Manjunath N.
Sprunger Suzanne A.
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