Patent Application
20050266412
This invention relates generally to the field of proteins and more specifically to the field of infectious proteins and methods of amplifying and detecting such proteins.
Some proteins can change from a normal configuration to an abnormal disease configuration. One such protein is PrP protein which can have a PrPC normal configuration and a PrPSc disease configuration. The disease configuration is often referred to as a prion.
Prions are infectious pathogens that cause invariably fatal prion diseases (spongiform encephalopathies) of the central nervous system in humans and animals. Prions differ significantly from bacteria, viruses and viroids. The dominating hypothesis is that no nucleic acid is necessary to allow for the infectivity of a prion protein to proceed.
A major step in the study of prions and the diseases they cause was the discovery and purification of a protein designated prion protein [Bolton, McKinley et al. (1982) Science 218:1309-1311; Prusiner, Bolton et al. (1982) Biochemistry 21:6942-6950; McKinley, Bolton et al. (1983) Cell 35:57-62]. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPC is encoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell 46:417-428] and when PrPC is expressed it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases results from the transformation of the normal form of prion protein (PrPC) into the abnormal form (PrPSc). There is no detectable difference in the amino acid sequence of the two forms. However, PrPSc when compared with PrPC has a conformation with higher β-sheet and lower α-helix content [Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1993) J Biol Chem 268:20276-20284]. The presence of the abnormal PrPSc form in the brains of infected humans or animals is the only disease-specific diagnostic marker of prion diseases.
PrPSc plays a key role in both transmission and pathogenesis of prion diseases (spongiform encephalopathies) and it is a critical factor in neuronal degeneration [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 103-143]. The most common prion diseases in animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr Top Microbiol Immunol 172:21-38]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Streussler-Sheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschler et al. (1992) N Engl J Med 326:444-449]. Initially, the presentation of the inherited human prion diseases posed a conundrum which has since been explained by the cellular genetic origin of PrP.
Prions exist in multiple isolates (strains) with distinct biological characteristics when these different strains infect in genetically identical hosts [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 165-186]. The strains differ by incubation time, by topology of accumulation of PrPSc protein, and in some cases also by distribution and characteristics of brain pathology [DeArmond and Prusiner (1997) Greenfield's Neuropathology, 6th Edition:235-280]. Because PrPSc is the major, and very probably the only component of prions, the existence of prion strains has posed a conundrum as to how biological information can be enciphered in a molecule other than one comprised of nucleic acids. The partial proteolytic treatment of brain homogenates containing some prion isolates has been found to generate peptides with slightly different electrophoretic mobilities [Bessen and Marsh (1992) J Virol 66:2096-2101; Bessen and Marsh (1992) J Gen Virol 73:329-334; Telling, Parchi et al. (1996) Science 274:2079-2082]. These findings suggested different proteolytic cleavage sites due to the different conformation of PrPSc molecules in different strains of prions. Alternatively, the observed differences could be explained by formation of different complexes with other molecules, forming distinct cleavage sites in PrPSc in different strains [Marsh and Bessen (1994) Phil Trans R Soc Lond B 343:413-414]. Some researchers have proposed that different prion isolates may differ in the glycosylation patterns of prion protein [Collinge, Sidle et al. (1996) Nature 383:685-690; Hill, Zeidler et al. (1997) Lancet 349:99-100]. However, the reliability of both glycosylation and peptide mapping patterns in diagnostics of multiple prion strains is currently still debated [Collings, Hill et al. (1997) Nature 386:564; Somerville, Chong et al. (1997) Nature 386:564].
A system for detecting PrPSc by enhancing immunoreactivity after denaturation is provided in Serban, et al., Neurology, Vol. 40, No. 1, Ja 1990. Sufficiently sensitive and specific direct assay for infectious PrPSc in biological samples could potentially abolish the need for animal inoculations completely. Unfortunately, such does not appear to be possible with current PrPSc assays—it is estimated that the current sensitivity limit of proteinase-K and Western blot-based PrPSc detection is in a range of 1 μg/ml which corresponds to 104-105 prion infectious units. Additionally, the specificity of the traditional proteinase-K-based assays for PrPSc is in question in light of recent findings of only relative or no proteinase-K resistance of undoubtedly infectious prion preparations [Hsiao, Groth et al. (1994) Proc Natl Acad Sci USA 91:9126-9130] Telling, et al. (1996) Genes & Dev.
Human transthyretin (TTR) is a normal plasma protein composed of four identical, predominantly β-sheet structured units, and serves as a transporter of hormone thyroxine. Abnormal self assembly of TTR into amyloid fibrils causes two forms of human diseases, namely senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) [Kelly (1996) Curr Opin Strut Biol 6(1):11-7]. The cause of amyloid formation in FAP are point mutations in the TTR gene; the cause of SSA is unknown. The clinical diagnosis is established histologically by detecting deposits of amyloid in situ in biopsy material.
To date, little is known about the mechanism of TTR conversion into amyloid in vivo. However, several laboratories have demonstrated that amyloid conversion may be simulated in vitro by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993) Biochemistry 32(45):12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun 197(2) 415-21]. The mechanism of conformational transition involves monomeric conformational intermediate which polymerizes into linear β-sheet structured amyloid fibrils [Lai, Colon et al. (1996) Biochemistry 35(20):6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxine or triiodophenol [Miroy, Lai et al. (1996) Proc Natl Acad Sci USA 93(26):15051-6].
In view of the above points, there is clearly a need for a specific, high flow-through, and cost-effective assay for testing sample materials for the presence of a pathogenic protein including transthyretin and prion protein.
In addition to PrP and TTR there are other proteins associated with other diseases.
The following is a non-limiting list of diseases with associated insoluble proteins which assume two or more different conformations.
It should be noted that the insoluble proteins listed above each include a number of variants or mutations which result in different strains which are all encompassed by the present invention. Known pathogenic mutations and polymorphisms in the PrP gene related to prion diseases are given below and the sequences of human, sheep and bovine are given in U.S. Pat. No. 5,565,186, issued Oct. 15, 1996.
When these proteins are present in very small amounts the individual does not exhibit symptoms of disease. It would be desirable to know that small amounts of the disease form of the protein are present if only to prevent passing the infection on to another individual. However, current assays can not, in general, detect the protein below a certain level. Thus, there is a need for a method whereby small amounts of infectious protein present in a sample can be amplified. The present invention provides such.
A method is disclosed whereby the amount of infectious protein present in a sample is amplified. The method comprises adding a recombinantly produced protein or portion thereof (corresponding to the protein to be amplified) to a sample which may contain the disease form of the protein to be amplified. Conditions promoting amplification are maintained (in vitro) over a limited period of time after which the sample is tested for the presence of the disease form of the protein. Infectious proteins being tested for are those generally associated with neurodegenerative diseases including but not limited to prion diseases, e.g. Parkinson's, and Alzheimer's. Thus, by detecting proteins associated with the disease in a sample it is possible to enhance the accuracy of diagnosing the disease in a patient from which the sample was extracted.
An aspect of the invention is that recombinantly produced proteins in a non-disease conformation can be used to increase the amount of a disease conformation of a protein in a sample.
Another aspect of the invention is that recombinantly produced portion(s) of a protein of interest can be added to a sample containing a disease conformation of that protein to increase the amount of the disease conformation of the protein in the sample.
Yet another aspect of the invention is that the recombinantly produced protein or portion thereof may be any animal protein, e.g. mammalian protein, e.g. human or cow protein that assumes both a normal and a disease conformation.
Still another aspect of the invention is that the protein amplification methodology can be used to prepare a sample for assaying in any type of assay by amplifying the protein of interest in a sample being tested.
Still yet another aspect of the invention is that it can be used on any type of sample including, brain tissue, nerve cells, muscle tissue, blood, cells and tissue used in transplantation, etc. in order to enhance the sensitivity of any assay used on such to detect infectious proteins.
These and other aspects, advantages, and objects of the invention will become apparent to those persons skilled in the art upon reading this disclosure in combination with the figures attached hereto.
The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:
Before the present protein amplification method and reagent used therewith are described, it is to be understood that this invention is not limited to particular embodiment or proteins described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates. otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of such proteins and reference to “the reagents” includes reference to one or more reagents and equivalents thereof known to those skilled in the art reading this disclosure, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
The present invention shows, for the first time, that it is possible to make an infectious protein in vitro in a cell free system. The proteins made have been shown to be infectious by inoculating transgenic mice with proteins produced. Because methods shown here “seed” the reagent with amyloid fibrils those skilled in the art reading this disclosure will understand that the “seeding” can be replaced with infectious proteins present in a sample to be tested and that the sample may be treated. When the sample contains the “seed” or infectious protein that protein will be amplified or produced many times over. Such amplification increases the sensitivity of assays to detect the presence of infectious proteins.
In it's simplest form the invention involves combining a reagent with a sample and maintaining the combination under conditions which allow for amplification of any infectious proteins present in the sample. The reagent is comprised of a recombinant protein, or a portion of a recombinant protein such as a significant C-terminal portion of a protein which corresponds to the amino acid sequence of the native protein being amplified. The method is carried out in vitro and in particular in a cell free system. The amplification is carried out and the assay is the run thereafter in a period of time less than 40 hours, preferably less than 20 hours, and may be in increments of 8, 7, 6, 5, 4, 3, 2, or 1 hour or less in order to obtain the desired amplification of any infectious protein in the sample. An important aspect of the invention is that the resulting amplified protein is shown to be infectious when used to inoculate an animal. Specifically, if the amplified protein is a human PrP protein and specifically a human prion when that human prion is used to inoculate a transgenic mouse which has a human PrP gene therein the mouse will become sick showing distinct evidence of a prion disease.
Although the invention is described here in connection with the PrP proteins and prion diseases those skilled in the art reading this disclosure will understand that the invention can be used in connection with proteins associated with other diseases wherein the protein is an infectious protein, i.e. a protein which assumes a first normal configuration and a second disease related conformation wherein both conformations have the same amino acid sequence. Examples of such diseases and their associated insoluble proteins are described in the background of this disclosure.
Specific methods of carrying out amplification using very specific reagents and conditions are described here. Those skilled in the art will understand that a degree of variation is permitted and that such is still within the scope of the present invention. Within the examples preparations are “seeded” with amyloid fibrils. However, to carry out the method of the invention on a sample the amyloid fibrils will be the infectious proteins present within the sample. If no infectious proteins are present then no amplification will occur in the time allowed for (see
The sample preparation methodology may involve concentration of any prions or other insoluble proteins which might be present in the sample. Sample concentration can be carried out by adding to the sample a binding agent such as phosphotungstic acid or a salt thereof which binds to the insoluble form of such proteins such as PrPSc. The binding agent is one such that when the binding agent to the protein alone. Thus, the combination can be subjected to a centrifuge in order to concentrate the protein bound to the binding agent and the concentrate can be tested. Such methods of sample preparation are described within U.S. Pat. No. 5,977,324 issued Nov. 2, 1999. It will also be understood by those skilled in the art reading this disclosure that the sample may be subjected to other processing such as by contacting the sample with enzymes which will cleave away portions of the insoluble protein leaving only a distinct insoluble core, e.g. PrP27-30 as described within U.S. Pat. No. 5,977,324.
In addition to sample preparation methods as described above those skilled in the art reading this disclosure will understand that certain samples require very specific preparation. For example, it is difficult to detect insoluble proteins such as insoluble PrP proteins within blood. To obtain a positive reading in a blood sample which has infectious proteins therein it is generally necessary to allow the blood to clot and then separate the serum away from the clotted blood. The serum is then contacted with a complexing agent of the type as described above in order to form a complex an then concentrate the complex to carry out the assay. Methods of this type are described within U.S. Pat. No. 6,166,187 issued Dec. 26, 2000.
The methodology of the present invention does not merely produce proteins. An important aspect of the invention is that the proteins produced are “infectious” in that they are capable of causing disease in an animal. Infectious proteins produced in accordance with the methodology disclosed herein have been tested in transgenic animals in order to confirm that they are infectious. Others can confirm such by using transgenic animals such as the transgenic mice disclosed and described within U.S. Pat. No. 5,792,901. Further, those animals can be used in controlled studies using standard prion preparations of the type described within U.S. Pat. No. 6,020,537 issued Feb. 1, 2000.
Once the method of the invention has been carried out in order to produce infectious proteins, i.e. amplify the amount of infectious proteins in the sample the sample can be further prepared as described above and can be assayed for the presence of such infectious proteins. It is possible to use essentially any assay known to test for infectious proteins such as the conformation dependent immunoassay (CDI) of the type described within U.S. Pat. No. 5,891,641 issued Apr. 6, 1999. It is also possible to confirm the presence of the infectious proteins using antibodies such as the antibodies disclosed and described within U.S. Pat. No. 5,846,533 issued Dec. 8, 1998. It may be desirable to use specific antibodies such as when assaying bovine brain for prions which infect cows. A specific antibody useful for assaying ungulates is disclosed within U.S. Pat. No. 6,537,548 issued Mar. 25, 2003.
To demonstrate the amplification methodology of the present invention an experiment was carried out to refold wt MoPrP(89-230) into amyloid fibrils and bioassay those fibrils in mice expressing the corresponding PrP. The PrP amyloid represents a limited subset of β-rich PrPs, all of which are infectious. It is important to note that PrP amyloid deposition is a nonobligatory constituent of prion diseases (S. B. Prusiner et al., Cell 63, 673-686 (1990)), in contrast to some other disorders in which amyloids seem to be constant features (C. M. Dobson, Nature 426, 884-890 (2003)).
Because PrP 27-30 polymerizes into amyloid fibrils (S. B. Prusiner et al., Cell 35, 349-358 (1983); M. P. McKinley et al., J. Virol. 65, 1340-1351 (1991)) and full-length PrPSc does not, N-terminally truncated MoPrP composed of residues 89-230 was expressed in E. coli. This protein, denoted recMoPrP(89-230) or recMoPrP(Δ23-88), was purified to homogeneity and folded into a β-sheet-rich state that assembled into amyloid fibrils which fibrils are shown in the electron micrograph of
After producing both seeded and unseeded amyloid fibrils composed of recMoPrP(Δ23-88), Tg(MoPrP, Δ23-88)9949/Prnp0/0 mice, hereafter referred to as Tg9949 mice were inoculated. The Tg9949 mice express MoPrP(Δ23-88) at a level 16-fold greater than SHaPrP in Syrian hamsters (S. Supattapone et al., J. Virol. 75, 1408-1413 (2001)). The Tg9949 mice received intracerebrally either unseeded or seeded amyloid preparations and were followed for clinical signs of nervous system dysfunction. All of the mice developed neurologic disease between 380 and 660 days after inoculation (see
aAll transgenes are expressed in Prnp0/0 mice. Expression levels of PrPC relative to normal SHaPrP levels in hamster brain were determined by immunoblots of serially diluted brain homogenates.
bNumber of animal developing prion disease/total number.
*Tg9949 mice did not show any signs of neurologic dysfunction over 670 days of age, at which time they were sacrificed. An additional uninocluated Tg9949 mouse was sacrificed at 580 days of age and failed to show any vacuolation or PrP deposits on neuropathologic evaluation or any protoease-resistant PrP on Western blotting.
The mice inoculated with seeded amyloid exhibited shorter incubation times compared to those with unseeded amyloid. Seven uninoculated Tg9949 mice remained healthy for 670 days and were sacrificed after the last amyloid-inoculated Tg9949 mice developed illness. In earlier studies, uninoculated Tg9949 mice lived for more than 500 days without any signs of neurologic dysfunction (S. Supattapone et al., J. Virol. 75, 1408-1413 (2001)).
The shortest incubation time for a Tg9949 mouse inoculated with seeded amyloid was 382 days compared to 474 days for a Tg9949 mouse inoculated with unseeded amyloid. Western blot analysis of brain homogenates of these two mice revealed that the Tg9949 mouse inoculated with seeded amyloid had more protease-resistant PrP than the brain of the unseeded amyloid-inoculated mouse (see
Whether the different incubation times and diverse biochemical profiles reflect higher levels of PrPSc in the seeded amyloid compared to the unseeded or the creation of two different prion strains remains to be established. No protease-resistant PrP was found by Western blotting in the brain of an uninoculated Tg9949 mouse sacrificed at 580 days of age (
Neuropathological examination of Tg9949 mice inoculated with seeded synthetic prions revealed extensive vacuolation with associated gliosis in the cerebellum, hippocampus, brainstem and white matter (
Prions in the brains of Tg9949 mice that had been inoculated with seeded amyloid were designated “synthetic mammalian prion strain 1,” or SMP 1. Serial transmission of SMP1 prions from Tg9949 mice to wt FVB and Tg(MoPrP-A)4053 mice gave mean incubation times of 154 and 90 days, respectively (see
Well-defined PrP amyloid plaques as well as numerous, densely packed, finely granular PrPSc deposits were identifiable in second passage in both FVB (
The present invention is directed at producing synthetic prions in vitro using the formation of PrP amyloid as a surrogate marker for the folding of MoPrP(89-230) into a biologically active conformation. The rapidity and ease of measuring thioflavin T binding that reflects amyloid formation (J. H. Come, P. E. Fraser, P. T. Lansbury, Jr., Proc. Natl. Acad. Sci. USA 90, 5959-5963 (1993); H. LeVine, Protein Sci. 2, 404410 (1993)) facilitate at the ability to determine conditions under which recMoPrP(89-230) assembles into amyloid fibrils (I. V. Baskakov, G. Legname, M. A. Baldwin, S. B. Prusiner, F. E. Cohen, J. Biol. Chem. 277, 21140-21148 (2002)). The results provided here show that such fibrils harbor detectable levels of prion infectivity making it possible to draw a series of conclusions about mammalian prions that were previously elusive.
First, the results provided here show that the prion protein is both necessary and sufficient for infectivity; prions are infectious proteins.
Second, neither the Asn-linked oligosaccharides nor the glycosylphosphatidylinositol anchor are required for prion infectivity since the recMoPrP(89-230) used in the experiments described here contains neither of these posttranslational modifications (A. Taraboulos et al., Proc. Natl. Acad. Sci. USA 87, 8262-8266 (1990); S. J. DeArmond et al., Neuron 19, 1337-1348 (1997); P. Gambetti, P. Parchi, N. Engl. J. Med. 340, 1675-1677 (1999); J. A. Mastrianni et al, N. Engl. J. Med. 340, 1630-1638 (1999)).
Third, the biological information carried by distinct strains of prions resides in PrPSc. Moreover, variations in PrP glycosylation are not required for prion diversity.
Fourth, the spontaneous formation of prions, which is responsible for sporadic forms of prion disease in livestock and humans, can occur in any mammal expressing PrPC.
The results provided here show that prion diseases are disorders of protein conformation in which PrPC and PrPSc represent distinct structural states. Previous difficulties in creating in vitro infectious prions from recPrPs enriched for β-structure may be due the tendency of mammalian PrPs to fold into biologically irrelevant β-rich isoforms. Although the strategy used in the experiments described here may appear rather straightforward in retrospect, the use of recombinant PrP in the method described here eluded researches for many years.
From Tg mouse studies, it is well established that templates improve the likelihood of forming an infectious β-rich isoform (S. Supattapone et al., J. Virol. 75, 1408-1413 (2001); S. B. Prusiner et al., Cell 63, 673-686 (1990)). The results provided here show that “seeded” amyloid fibrils exhibit shorter incubation times than their “unseeded” progenitor (see
A bona fide cell-free amplification system for infectious proteins such as prions would be valuable in assaying the safety of a range of foods including beef, lamb, pork and chicken as well as a biological material obtained from a patient to treat another patient such as blood, blood products, cells, tissues, organs, etc. Thus, these results have important implications for human health. The formation of prions from recPrP demonstrates that PrPC is sufficient for the spontaneous formation of prions, and thus, no exogenous agent is required for prions to form in any mammal. The results shown here provide an explanation for sporadic Creutzfeldt-Jakob disease for which the spontaneous formation of prions has been hypothesized (S. B. Prusiner, Annu. Rev. Microbiol. 43, 345-374 (1989)). Understanding sporadic prion disease is particularly relevant to controlling the exposure of humans to bovine prions (A. G. Biacabe, J. L. Laplanche, S. Ryder, T. Baron, EMBO Rep 5, 110-115 (2004); C. Casalone et al., Proc Natl Acad Sci USA 101, 3065-3070 (2004); Y. Yamakawa et al., Jpn J Infect Dis 56, 221-222 (2003)). That bovine prions are pathogenic for humans is well documented in the cases of more than 150 teenagers and young adults who have already died from prion-tainted beef derived from cattle with bovine spongiform encephalopathy (BSE) (R. G. Will et al., Lancet 347, 921-925 (1996); M. R. Scott et al., Proc. Natl. Acad. Sci. USA 96, 15137-15142 (1999); R. G. Will, M. P. Alpers, D. Dormont, L. B. Schonberger, in Prion Biology and Diseases S. B. Prusiner, Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2004) pp. 629-671). Moreover, the sporadic forms of Alzheimer's and Parkinson's diseases as well as amyotrophic lateral sclerosis and the frontal temporal dementias are the most frequent forms of these age-dependent disorders as is the case for the prion diseases (L. E. Hebert, P. A. Scherr, J. L. Bienias, D. A. Bennett, D. A. Evans, Arch Neurol 60, 1119-1122 (2003)). Important insights in the etiologic events that feature in these more common neurodegenerative disorders, all of which are caused by the aberrant processing of proteins in the nervous system, are likely to emerge as more is learned about the molecular pathogenesis of sporadic prion diseases (S. B. Prusiner, N. Engl. J. Med. 344, 1516-1526 (2001)).
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Amyloid fibrils were formed upon incubation of recMoPrP(89-230) (0.6 mg/ml) at 37° C. in 3 M urea, 0.2 M NaCl, 50 mM Na-acetate buffer, pH 5.0 as previously described (I. V. Baskakov, G. Legname, M. A. Baldwin, S. B. Prusiner, F. E. Cohen, J. Biol. Chem. 277, 21140-21148 (2002)). The kinetics of fibril formation were monitored using a thioflavin T binding assay (H. LeVine, Protein Sci. 2, 404-410 (1993)). Inocula were prepared by dialysis of the fibrils in PBS buffer, pH 7.2 for two days. Approximate concentration of recMoPrP(89-230) in the inocula was 0.5 mg/ml.
To obtain the data for the graph of
The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.
The United States Government may have certain rights in this application pursuant to Grant Nos. AG02132, AG10880 and AG021601 awarded by the National Institutes of Health.
