Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-07-09
2004-06-15
Clark, Deborah J. R. (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C530S350000, C530S380000
Reexamination Certificate
active
06750025
ABSTRACT:
BACKGROUND OF THE INVENTION
Native prion protein (PrP
c
) is widely distributed throughout nature and is particularly well conserved within mammals (Caughey and Chesebro,
Trends Cell Biol
. 7:56-62 (1997)). In man it is transcribed from a gene present on chromosome 20 (Sparks, et al.
Proc. Natl. Acad. Sci. USA
83:7358-7362 (1986)). It is the conversion of the native PrP
c
protein to the infectious PrP
sc
protein which leads to the propagation of diseases such as kuru and Creutzfeld-Jakob disease in man, scrapie in sheep, bovine spongiform encephalopathy, transmissible mink encephalopathy, and wasting disease in deer and elk. The PrP
c
gene contains one open reading frame with end terminal sequencing of the expressed protein beginning early (position 3) in the open reading frame. In scrapie infected hamsters, the first amino acid of PrP
c
is lysine at position 3, and for the infectious PrP
sc
protein it is lysine at position 15 (Turk, et al.
Eur. J. Biochem
. 176:21-30 (1988)). In its natural form, the protein has a significant portion of random loop structure at the amino terminal which includes the octapeptide repeat sequences of Gly-Tyr-Gly-Gln-Pro-His-Gly-Gly (SEQ. ID NO: 1) (Miura, et al. FEBS Lett. 396:248-252 (1996)). Four repeats of this sequence are present between residues 53 and 91 of the open reading frame in the human protein. Some species, such as cattle, may have more repeats of this sequence. The protein in amyloid fibrils sometimes designated PrP 27-30 may lack this sequence while it is also removed experimentally through the action of proteinase K. The octapeptide sequence is highly conserved among mammalian prion proteins implying some structural and functional roles of the octapeptide. Being rich in glycine and praline neither alpha helix nor beta sheet are predicted for this region though it is able to assume structure in the presence of copper (Miura, et al. FEBS Lett. 396:248-252 (1996)). Prion protein is able to form a single intramolecular disulphide found between Cys 179 and 214. The translated protein is modified by glycosylation at two sites. In the hamster protein they are at positions 183 and 197 (Rogers, et al. Glycobiol. 1:101-109 (1990)). The expressed protein is localized to the outer surface of cell membranes by a glycoinositol phospholipid linked through ethanolamine attached to serine at position 231. It is a conformationally altered form of PrP
c
that is believed to be responsible for prion diseases. It is postulated that PrP
c
is converted into the infectious PrP
sc
protein through a posttranslational process during which it acquires a high &bgr;-sheet content (K. M. Pan, et al.
PNAS U.S.A
. 90:10962 (1993)). Sheet modification is believed to start at residues close to the carboxy terminal side of amino acid 100. It is thought likely that a non-infectious protein must interact directly with an infectious prion protein for this conversion to take place, and it has been proposed that sites exist on the central portion of the PrP molecule that allow specific binding between PrP
c
and PrP
sc
as a first step in the conversion of PrP
c
to PrP
sc
(Chabry, et al. 1998). It is not known how prion proteins interact together though it is considered likely that an additional protein or “chaperone” is required. This structural change of the factor is accompanied by changes in the properties of the protein. PrP
sc
protein is not soluble in nondenaturing agents, whereas PrP
c
is (R. K. Meyer, et al.,
PNAS U.S.A
. 83:2310 (1986)); and PrP
sc
protein is partially resistant to digestion by proteases, whereas PrP
c
is highly digestable (B. Oesch, et al.,
Cell
40:735 (1985)).
Detection of prion protein is usually accomplished by the use of monoclonal antibodies, of which 3F4 is currently used extensively (Kascsak, et al. 1987). Antibody 3F4 recognizes the peptide sequence Met-Lys-His-Met (SEQ ID NO: 4) of the native conformation of PrP
c
and also Met-Lys-His-Met (SEQ ID NO: 4) of denatured, but not fibrils of PrP
sc
. Consequently, in order to detect the presence of aggregates of PrP
sc
, the proteins must first be denatured. A monoclonal specific for the resistant form has been reported, but its utility has not been verified. Other monoclonal antibodies have been reported, but in general their specificity and affinity for the prion protein are poor. The major problem in generating a sufficient immunological response to provide useful antibodies is the fact that the structure of prion proteins is highly conserved in animals, so immunogens comprising prions from different species are recognized by the immunized animal as host proteins. The amino terminal including the Gly-Tyr-Gly-Gln-Pro-His-Gly-Gly (SEQ ID NO: 1) repeat sequence of the protein is present as a random coil and presumably lacks a defined structure. Consequently, it will not be presented as a defined epitope.
Thus, a great need remains for a method of detection of PrP
sc
protein in a sample which overcomes the problems of the prior art methods, such as the methods using antibodies. Specifically, a method is needed that allows for the direct detection of PrP
sc
protein in a sample which is specific and does not require manipulation of the protein before detection. A method of detection of the PrP
sc
protein which overcomes the problems of the prior art will be extremely valuable for detecting and diagnosing prion diseases in a subject.
Finally, a need also remains for a way to bind prion proteins to each other to study their interaction. This would be an extremely valuable research tool for designing molecules that will interfere with the binding of prion proteins together and thus prevent the conversion of a native prion protein to one that is infectious.
SUMMARY OF THE INVENTION
The present invention provides a method of detecting prion protein in a sample. Using the method provided herein, all variants and strains of prion protein can be detected. The method comprises contacting the sample with an agent that binds to the amino acid sequence Gln-Pro-His of prion protein and detecting the agent bound to the prion protein. In order to detect the agent, the agent may be bound to a detectable marker, such as a radiolabel or an enzyme. In a specific embodiment of the invention, the agent is streptavidin which binds tightly and specifically to the Gln-Pro-His sequences of prion proteins.
The method of detection of prion proteins provided by the present invention overcomes the problems presented by the prior art methods of detection using antibodies. The method provided herein does not require denaturation of the prion proteins in order to detect them. Furthermore, unlike antibodies to a protein which vary in specificity from strain to strain, and most certainly from species to species, the novel method provided herein utilizes a highly conserved amino acid sequence of the prion protein as a binding site for agents such as streptavidin to bind to and allow for detection. The amino acid sequence utilized by the present invention is found to be highly conserved throughout all prion protein variants and strains, as well as throughout all species.
Also provided by the present invention is a method of isolating prion protein in a sample by contacting the sample with an agent that binds to the amino acid sequence Gln-Pro-His of prion protein and isolating the agent bound to the prion protein.
A method of diagnosing a prion disease in a subject is also provided by the present invention. This method comprises contacting a biological sample taken from the subject with an agent that binds to the Gln-Pro-His sequence of prion protein, then detecting the agent in the sample. The prion diseases which may be diagnosed using the method of the present invention are Creutzfeld-Jakob disease, Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia, scrapie, bovine spongiform encephalopathy (mad cow disease), transmissible mink encephalopathy, feline spongiform encephalopathy, exotic ungulate encephalopathy, and chronic wasting disease.
The present invention also provides a method for
Hammond David J.
Medina Emma L.
Amster Rothstein & Ebenstein
Baker Anne-Marie
Clark Deborah J. R.
V.I. Technologies, Inc.
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