Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
1999-01-25
2001-10-30
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C436S547000, C436S548000, C530S387900, C530S388100, C530S388200, C530S389100, C530S413000
Reexamination Certificate
active
06309863
ABSTRACT:
BACKGROUND OF THE INVENTION
Protein phosphorylation is important in the regulation of a wide variety of cellular processes. Regulation of protein activity by phosphorylation of serine, threonine, and tyrosine residues is highly utilized. Histidine, arginine, and lysine residues on proteins are also phosphorylated by cellular processes, but the significance is unknown due to the difficulty of studying these highly unstable modifications. The detection and quantitation of changes in the phosphorylation state of a protein is of great utility in the study of its functional significance.
Standard methods for measuring the state of protein phosphorylation typically involve prelabeling of the added phosphate moiety by incorporation of a radioactive isotope of phosphorous (as a phosphate). Such phosphorylation assays suffer from several methodological pitfalls, including health risks and disposal problems associated with the high amounts of [
32
P]Pi required for the prelabeling experiments, the bother of working with regulated substances, and a lack of site specificity when several sites are phosphorylated in one protein or peptide moiety. As a result of these drawbacks, immuno-chemical based methods for detecting protein phosphorylation state are increasing in popularity. The degree of sensitivity and selectivity achievable with immuno-chemical methodology makes it an attractive alternative (Matsui et al.,
J. Cell. Bio
. 140: 647-657 (1998); Conrad et al.,
Hybridoma
16: 167-173 (1997)).
Phosphorylation state-dependent monoclonal antibodies specific for a variety of cytoskeletal proteins have been produced and characterized. These antibodies were isolated by immunization protocols in which the specific targeting of phosphorylated epitopes was not the primary objective. More recently, small synthetic phosphopolypeptides have been used to improve the chance of targeting antibody production to epitopes on the phosphorylation sites (Sakaguchi et al.,
Genes and Dev
. 12: 2831-2841 (1998); Matsui et al.,
J. Cell Bio
. 140: 647-657 (1998); Chen et al.,
FASEB J
. 2: A550 (1988); Czernik et al.,
Methods in Enzymology
201: 264-283 (1991)). While more direct, this method still suffers from the limitation of rapid dephosphorylation of the polypeptide antigen upon immunization which reduces the titer of phospho-specific antibodies. This is particularly a problem when using antigen containing phosphoserine and phosphothreonine, both of which usually are considerably less stable than phosphotyrosine.
SUMMARY OF THE INVENTION
The present invention provides methods for generating antibodies which specifically react to a polypeptide phosphorylated at a particular amino acid. Methods for generating both monoclonal and polyclonal antibodies are provided. The method involves providing a polypeptide which has an incorporated mimetic of the phosphorylated amino acid residue. The mimetic has antigenic determinants also present on the naturally phosphorylated amino acid. The polypeptide antigen is used by standard methods to generate either monoclonal or polyclonal antibodies which cross-react with the natural phosphorylated polypeptide, and specifically recognize a specific phosphorylation state of the polypeptide.
In one embodiment, the mimetics contains a non-hydrolyzable linkage between the carbon atom and the phosphorous atom (of the phosphate group). In a preferred embodiment, this linkage is a CF
2
group. Incorporation of this linkage group into phosphoserine produces the mimetic F
2
Pab. F
2
Pab is used in place of phosphoserine in a polypeptide sequence derived from p53 to produce antibodies which recognize a specific phosphorylation state of p53. In another embodiment, the CF
2
linkage group is incorporated into phosphothreonine to produce the mimetic F
2
Pmb. In another embodiment the CF
2
linkage is incorporated into phosphotyrosine to produce the mimetic F
2
Pmp.
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patent: 5814459 (1998-09-01), Montminy
Sakaguchi et al.,Genes and Dev. 12: 2831-2841 (1998).
Matsui et al.,J. Cell Biol. 140: 647-657 (1998).
Burke et al.,Biochem. Biophys. Res. Commun. 204: 129-134 (1994).
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Otaka et al.,Tetrahedron Letters 36: 927-930 (1995).
Akamatsu et al.,Bioorg.&Med. Chem. 5: 157-163 (1997).
Conrad et al.,Hybridoma 16: 167-173 (1997).
Matsumara et al.,J. Cell Biol. 140: 119-129 (1998).
Ginty, et al., “Regulation of CREB Phosphorylation in the Suprachiasmatic Nucleus by Light and a Circadian Clock”,Science260, 1993, 238-241.
Ginty, et al., “Nerve Growth Factor Activates a Ras-Dependent Protein Kinase that Stimulates c-fos Transcription via Phosphorylation of CREB”,Cell77, 713-725.
Miyoshi, et al., “Practical Phosphopeptide Synthesis Using Dimethylphosphono Amino Acids (No. 2)”,Peptide Chemistry33, 25-28.
Shieh, et al., “DNA Damage-Induced Phosphorylation of p53 Alleviates Inhibition by MDM2”,Cell91, 325-334.
Siliciano, et al., “DNA Damage Induces Phosphorylation of the Amino Terminus of p53”,Genes&Development11, 3471-3481.
Anderson Carl W.
Appella Ettore
Sakaguchi Kazuyasu
Bogosian Margaret C.
Brookhaven Science Associates
Saunders David
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