MXPA99004171A - Soluble proteins - Google Patents

Abstract

The invention includes deleting codon segments from DNA expressing a native protein in order to obtain a shorter, soluble protein which mimics characteristics of an insoluble native protein. Soluble proteins of the invention are characterized by:(1) having less amino acids than the full length native protein;(2) having a higher degree of solubility than the native protein;(3) retaining the basic biological characteristics of the native protein such as (a) not being subject to enzymatic digestion and (b) causing disease. Soluble proteins of the invention are obtained by providing a DNA sequence which encodes a native protein and systematically removing codons, making copies of the shortened versions of DNA which are then expressed to provide the shortened proteins. The shortened proteins are then tested for solubility. Soluble proteins are then further tested to confirm that they retain the biological characteristics of the native protein.

Description

SOLUBLE PROTEINS I I DESCRIPTION OF THE INVENTION | This invention relates generally to the field of molecular genetics and more particularly to methods for making '5 soluble insoluble proteins and soluble proteins and derivatives thereof. I A number of proteins found in 'natural way that they are insoluble and be related to a state I of particular disease. Examples include plates I 10 protein ß amyloids associated with Alzheimer's disease, Lewi bodies or Pic bodies associated with diseases of Parkinson's and Pick's and prion proteins (ie, PrPS) ! associated with Creutzfeldt-Crab disease (CJD) and others 'neurogenic disorders. The PrP gene of mammals company I 15 a protein which can be the soluble PrP ° form, of non-disease or be converted to insoluble PrPSc or I-form! disease. There are a number of systematic diseases that | Associated with protein aggregation including multiple myeloma, thyroid carcinomas and congophilic angipathies. I 20 The importance of understanding the conversion of PrP = in | in PrPSc has been highlighted by the possibility that prions I I bovines have been transmitted to humans who develop Creutzfeldt-variab variant disease (CJDv), G. Chazot, et al., Lancet 347, 1181 (1996); R.G. Will, et al., Lancet 347, 921-925 (199S). The most recent studies have shown that N-termination of PrPSc can be -truncated without loss of squeegee infectivity, S.B. Prusiner, et al., Biochemistry 21, 6942-5950 (1982)); S.B. Prusiner, et al., Cell 38, 127-134 (1984) and correspondingly, the truncation of the N terminus of PrPs = still allows its conversion into PrPS. M. Rogers, et al., Proc. Nati Acad. Sci. USA 90, 3182-3186 (1993). However, any form of PrPSc (ie, the protein form which causes the disease) is also insoluble. The insolubility of the protein makes the | 10 analyze and greatly hinder efforts to generate an antibody which binds to the protein. Further, The soluble form of the protein (ie, PrPc) is copocida '(1) to be digestible with enzymes that do not digest PrPSc and (2) I do not cause disease. | 15 The insolubility of certain proteins associated with I diseases have made it difficult to carry out the structural analysis of such proteins or to generate specific antibodies for these proteins. The present invention provides soluble forms of such proteins. This could be obtained in different ways such as (i) systematically removing ^ segments of DNA that encode the protein 'native or (2) adding DNA and then expressing the modified ij protein or (3) chemically modifying the insoluble protein I such as adding amino acids with side chains I 25 hydrophilic or other hydrophilic groups. The protein The resulting (1) is soluble, (2) it can not be digested by enzymes that digest the native soluble form, and (3) it retains its biological function (for example, it maintains anomalous properties associated with disease). The synthetic soluble forms of these proteins are useful for generating antibodies which selectively bind the modified soluble proteins as well as the native insoluble proteins. Soluble proteins can also be used to evaluate drugs with respect to their effect on the protein in terms of the ability of the drugs to eliminate the abnormal properties of the proteins, associated with the disease. A soluble form of an insoluble prptein that occurs naturally, such as a soluble form of native PrPsc, is produced by the process described in the prepente. Although other processes can be used to obtain a soluble form of proteins that are insoluble in their native form, this invention encompasses soluble proteins of the type described herein produced by other methods. The general method involves first determining the DNA sequence that encodes a protein that is either expressed in an insoluble form or then converted to an insoluble protein, in which the insoluble protein is associated with a disease of the nervous system. copies of the DNA sequence. By using the individual copies or groups of copies, a portion or portions of the DNA is removed. The elimination is done, of course, in codon segments, whereby a plurality of different DNA sequences are provided which are different from each other in view of the different codon sequences deleted. These different shortened sequences are then expressed and a plurality of different proteins are obtained. The different proteins obtained are tested for solubility. Proteins are then tested which are soluble with respect to another activity such as if the protein can be digested by enzymes which normally do not digest the insoluble protein. If the protein is both soluble and resistant to digestion, it can also be tested for biological activity. For example, the protein can be used to inoculate an animal which can normally develop disease when inoculated with the insoluble native form of the protein. If the inoculated animal develops the disease, then the protein is part of the present invention and characterized as (1) soluble; (2) not subject to enzymatic digestion; (3) biologically active, for example, with respect to its ability to cause disease and / or generate antibodies that bind to the insoluble native protein, One object of the invention is to provide soluble forms of proteins that are insoluble in nature where the soluble forms maintain biological characteristics of the insoluble native form. - A more specific object is to provide a form soluble of PrP (that is to say, a soluble prion) which maintains the characteristics that cause the biological disease of prpsc inso? ujD e / native and which generates antibodies with bound PrPSc. Another object of the invention is to provide a method for systematically removing or adding segments of DNA to obtain shorter or larger sequences which express a soluble form of a protein which causes disease which is insoluble in a native form. Another object is to provide proteins which convert native insoluble proteins to a soluble form which is then degraded thereby providing useful proteins in the treatment of diseases associated with insoluble proteins. Another object of the invention is to provide antibodies and methods for producing such antibodies using the soluble proteins of the invention, wherein the antibodies bind to the native insoluble form of the protein. Another object of the invention is to provide a method for producing a soluble form of a protein that is capable, in vivo, of binding to a protein aggregation test form resulting in a complex which delays, inveases or completely stops aggregation of protein with a disease.

Another object is to provide assay devices that are comprised of carriers having antibodies bound to their sce where the antibodies are generated using the soluble proteins of the invention. An advantage of the invention is that the soluble proteins are more easily subject to structural analysis and facilitate the ability to generate antibody compared to insoluble proteins. A feature of the invention is that the soluble proteins of the invention maintain important biological functions of native insoluble proteins. Another feature is that the soluble protein that has abnormal properties associated with disease can be used to evaluate drugs which convert the protein to its non-disease conformation. These and other objects, advantages and features of the invention will become apparent to those skilled in the art after reading this description. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a map of regions of protein secondary structure expressed by a PrP gene in terms of the requirement for each region of the protein to support the formation of PrPSc. Before the soluble proteins of the present and methods to do the same, it is understood that this invention is not limited to the proteins, methods, antibodies or particular processes described since such may, of course, vary. It is also 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. It should be noted that as used in this specification and the appended claims, the singular forms "a" and "," and "the," include plural references unless the context clearly dictates otherwise. Thus, for example, the reference to "a protein" includes mixtures and large numbers of proteins, the reference to "an antibody" includes large numbers of antibodies and mixtures thereof, and the reference to "the method" includes one or more methods or steps of the type described herein. The publications discussed herein are provided for description only prior to the date of filing the present application. Nothing herein is constructed as an admission that the present invention is not entitled before the fesha of such publication by virtue of the foregoing invention. Unless otherwise defined, all technical and scientific terms in the present have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

Although any methods and materials, similar or equivalent to those described herein, may be used, In the practice or test of the present invention, preferred methods and materials are described herein. All publications cited herein are incorporated herein by reference for the purpose of describing specific aspects of the invention for which the publication with connection. j DEFINITIONS The term "soluble" must mean a property I of a compound with respect to its ability to mix ! I in a liquid and specifically must mean its ability to dissolve in a liquid such as water, an aqueous salt, a mixed solvent or detergent solution to form a homogeneous mixture, that is, a solution and not simply a suspension. The solubility of a protein for the present invention can be defined using a variety of different parameters and those parameters may change depending on the particular protein whose solubility is being measured. In general, the insoluble native proteins which are associated with disease states are insoluble in water, salt solutions and mild detergents (ie, detergents that do not denature the protein) while the Soluble proteins of the invention are soluble in one or all of these. In addition, the soluble proteins of the invention can be placed in solution and subjected to 100,000 g for one hour and will remain in the supernatant. The PrPSc protein of the present invention is soluble in an aqueous solution of a mild detergent which does not denature the protein or the native PrPSc. When PrPs = is placed in aqueous solution of a mild detergent and subjected to 100,000 g in a centrifuge for one hour the PrPSc protein remains in the supernatant. A variety of different detergents can be used when checking the solubility of the modified protein. A suitable example of the detergent compositions includes a combination of sodium deoxycolate; 0.5% Triton X-100 and Sarcosil in an amount of 0.5%, 1.0%; or 2.0% as necessary. The protein PrPs = is soluble in any of the three concentrations of Sarcosil. In addition to including a detergent within the solvent it is preferable to include a salt and maintain the pH in the range of 5 to 9, more preferably 6 to 8 and still more preferably about 7.5 Examples of salts that can be used include 10 mM Tris-HCl a pH of 7.5; 100 mM NaCl (sodium chloride); or 1 mM EDTA. Other salts may also be used within the concentrations within the range of 1 mM to 100 mM. The term "insoluble" must be used in the present to describe a characteristic of a compound, and specifically a characteristic of a protein with respect to its ability not to mix with a liquid and not dissolve within a liquid in order to form a homogeneous mixture and specifically not to dissolve in water or an aqueous salt or detergent solution. Insoluble native proteins of the type described herein should be designated insoluble when the protein is not in the supernatant after subjecting the protein to 100,000 g for one hour when the protein is in water, water and a salt, water and a detergent which does not denature the protein or water, salt and a detergent which does not denature the protein. The terms "digestible" and "subject to enzymatic digestion" and the like are used interchangeably herein and should mean that a protein is cleaved by an enzyme in two or more pieces and preferably cleaved in a plurality (ie, three or more pieces). ) when contacted with the enzyme under conditions in which the enzyme can normally be expected to cleave a protein. The particular enzyme or enzymes used, the length of time the enzyme is contacted with the protein and other conditions such as temperature and presure will vary depending on the particular-modified protein being tested. When digestion tests are carried out on proteins expressed by modifying the PrP gene, it is preferable to use protease K and put K protease in contact with the protein.

For a period of time in the range of approximately 10 (minutes to 2 hours, more preferably 20 minutes to 1 hour and i still more preferably approximately 30 minutes to an i, temperature in the range above 0 ° C and less than 100 ° C more preferably 15 to 45 ° C and still more preferably I about 37 ° C at atmospheric pressure. If the protein is ! denatured when exposed to protease K by i about 30 minutes at about 37 ° C then the , protein does not possess the biological characteristics of the protein I I 10 Native PrPS which causes the disease. If the protein does not 1 is digested then the protein can be further tested for its ability to cause disease and be classified I as a protein of the present invention. 1 The terms "non-digestible" and "not subject to | 15 enzymatic digestion "is used interchangeably in the present j and should mean that when a protein is contacted With an enzyme the protein does not split. Specifically, the term should mean that the protein remains in one piece and does not divide when the enzyme is contacted with the protein under conditions that can normally result in the cleavage of the protein. The terms "PrP protein", "prp" and the like are used interchangeably herein and should mean both forms of infectious particle PrPSc known to cause disease (spongiform encephalopathies) in humans and animals and the non-infectious PrPSc form which, under the appropriate conditions, is converted to the infectious PrPSc form. The terms "prion", "prion protein" and "PrP ° protein" and the like are used interchangeably to refer to the infectious PrPSc form of a PrP protein and is a contraction of the words "protein" and "infection" and the particles they are well understood, if not exclusively, of PrPsc molecules encoded by a PrP gene. Prions are different from bacteries, viruses and viroids. Known prions include those that infect animals to cause squeegee, a transmissible, degenerative disease of the goat and goat nervous system as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies in cats. Four prion diseases known to affect the human are (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Strassler-Scheinker disease (GSS), and (4) fatal familial insomnia (FFI). ). As used in the present prion it includes all forms of prions that cause all or any of these diseases or others in any used animals - and in particular in domesticated humans and domestic animals. The term "PrP gene" is used herein to describe genetic material that expresses proteins as shown in Figures 2-4 of the Patent of the United States. ,565,186, filed October 15, 1996, and polymorphisms I and mutations such as those listed in the present under | the subtitle "Pathogenic Mutations and Polymorphisms", e 5, , 565,186. The term "PrP gene" generally refers to I 5 any gene of any species encoding any form of a prion protein. Some commonly known sequences of PrP are described in Gabriel et al., Proc. Nati i Acad. Sci. USA 89: 9097-9101 (1992) which is incorporated herein by reference to describe such sequences. He The PrP gene can be from any animal including the "host" and "test" animals described herein and any and all polymorphisms and mutations thereof, it is recognized that the terms include different other such Prp gene which is not yet They have been discovered. 15 The protein expressed for such a gene can assume either the PrPc (non-disease) form of PrPSc (disease). The terms "standardized prion preparation", "prion preparation", "preparation" and the like are used interchangeably herein to describe a The prion-containing composition (PrPs) in which the composition is obtained from mammalian brain tissue that contains substantially the same genetic material as | relates to prions, for example, brain tissue of a group j of mammals exhibiting signs of prion disease in which mammals (1) include a transgene as describes in the present; (2) have an ablative endogenous prion protein gene; (3) have a high copy number of prion protein genes of a genetically diverse species; or (4) are hybrids with an ablative endogenous prion protein gene and a prion protein gene of a genetically diverse species. The mammals from which the standardized prion preparations are obtained exhibit clinical signs of CNS dysfunction as a result of inoculation with prions and / or due to the development of the prion. disease due to its genetically modified construction, for example, high copy number of prion protein genes. The term "artificial PrP gene" is used herein to encompass the term "chimeric PrP gene" as well as Also other genes constructed in recombinant form which when included in the genome of a host animal (eg, a mouse) will lead the mammal to be susceptible to the infection of prions which naturally affect only one genetically diverse test mammal, for example, human, bovine or ovine. In general, an artificial gene will include the codon sequence of the PrP gene of the genetically altered mammal with one or more (but not all, and generally less than 40) codons of the natural sequence that is replaced with a different codon - preferably a codon I i 25 corresponding to a genetically diverse mammal (taj as a human) . The genetically altered mammal that is used to test prion samples that only infect the genetically diverse mammal. Examples of artificial genes are genes Mouse PrP that encode the sequence as shown in the Figures 2, 3 and 4 of 5, 565,186 with one or more different replacement codopes selected from the codons shown in these Figures for humans, cows, and goats and replacing mouse codons in the same relative position, 1 with the proviso that not all mouse codons are replaced with different human codons, from cows or goats.

I The artificial PrP genes can include not only codons of 1 genetically diverse animals but may include codons and codon sequences not associated with any PrP gene but which, when inserted into the animal, will lead the animal to being susceptible to infection with prions which can only normally infect a genetically diverse animal. The terms "chimeric gene", "chimeric PrP gene", "chimeric prion protein gene" and the like are used interchangeably here to mean a constructed gene artificially containing the codons of a host animal such as a mouse with one or more codons that are replaced with corresponding codons from a genetically diverse test animal such as a human, cow or pveja. I In a specific example, the chimeric gene is comprised of | 25 the start and end sequence (ie, the codes terminals N- and C-) of a PrP gene of a mammal of host species (eg, a mouse) and also containing a nucleotide sequence of a corresponding portion of a PrP gene of a second-species mammal (by example, a human). A chimeric gene, when inserted into the genome of a mammal of the host species, will lead the mammal to be susceptible to infection with prions which normally infect only mammals of second species. The MHu2M chimeric gene contains the start and stop sequence of a mouse PrP gene and a non-terminal sequence region which is replaced with a corresponding human sequence, which differs from a mouse PrP gene in a manner such that the protein expressed by it differs by nine residues. The term "genetic material related to prions" is proposed to cover any genetic material which effects the ability of an animal to become infected with prions. Thus, the term encompasses any "PrP gene", "artificial PrP gene", "chimeric PrP gene" or "ablative PrP gene" in which terms are defined herein as well as mutations and modifications thereof, which they effect the ability of an animal to become infected with prions. The standardized prion preparations of the invention are produced using animals which all have substantially the same genetic material in relation to to the prion in such a way that all animals will become infected with the same type of prions and will eventually show signs of infection at approximately the same time. The terms "host animal" and "host mammal" are used to describe animals which will have their genome genetically and artificially manipulated to include genetic material which is not naturally present within the animal. For example, the host animals include mice, hamsters and rats, which have their Ablative PrP genes ie, become inoperative. The host is inoculated with the prion proteins to generate antibodies. The cells that produce the antibodies are a source of the genetic material to make a phage library. Other host animals may have a natural gene (PrP) or μno which is altered by the insertion of an artificial gene or by the insertion of a native PrP gene from a genetically diverse test animal. The terms "test animal" and "test mammal" are used to describe the animal which is genetically diverse from the host animal in terms of the difference between the PrP gene of the host animal and the PrP gene of the test animal. The test animal can be any animal for which one wishes to run a test test to determine whether a given sample contains prions with which the test animal could generally be susceptible to the test. infection. For example, the test animal may be a human, cow, sheep, pig, horse, cat, dog or hen, and one may wish to determine whether a particular sample includes prions which could normally only infect the test animal. The terms "genetically diverse animal" and "genetically diverse mammal" are used to describe an animal which includes a PrP codon sequence native to the host animal which differs from the genetically diverse test animal by 17 or more codons, preferably 20 or more codons, and more preferably 28-40 codons. In this way, a mouse PrP gene is genetically diverse with respect to the PrP gene of a human, cow or sheep, but is not genetically diverse with respect to the PrP gene of a hamster. The terms "ablative prion protein gene", "altered PrP gene", "Ablative PrP gene" and the like are used interchangeably herein to mean an endogenous prion protein gene which has been altered (e.g., aggregated and / or removed nucledtides) in a form to carry gene to be non-operational. Examples of non-functional PrP genes and methods for making them are described in Büeler, H., et al. "Normal development of mice lacking the neuronal cell-surface PrP protein" Nature 356, 577-582 (1992) and Weisman (WO 93/10227 ). The methodology for making ablative a gene is taught in Capecchi, Cel 51: 503-512 (1987) all of which are Incorporate here for reference. Preferably both alleles of the genes are altered. The terms "hybrid animal", "transgenic hybrid animal" and the like are used interchangeably here 3 to mean an animal obtained from the cross-breeding of a first animal having an endogenous PrP gene ablativp, with a second animal, which includes either (1) a chimeric gene or an artificial PrP gene or (2) a gene PrP of a genetically diverse animal. For example, you get a hybrid mouse 3 by cross-breeding a mouse with an ablative mouse PrP gene with a mouse containing (1) human PrP genes (which may be present in high numbers of copies) or (2) chimeric genes. The term hybrid includes any I offspring of a hybrid that includes inbred offspring j 15 of two hybrids with the proviso that the resulting offspring are susceptible to infection with prions with normal infection only to genetically diverse species. A hybrid animal with prions can be inoculated and serve as a source of cells for the creation of hybridomas to make monoclonal antibodies of the invention. The terms "susceptible to infection" and "susceptible to infection with prions" and the like are used interchangeably herein to describe a transgenic or hybrid test animal of the invention which develops a ! 25 prion disease if inoculated with prions which they could normally only infect a genetically diverse test animal. The terms are used "to describe a transgenic or hybrid animal of the invention such as a Tg transgenic mouse (MHu2M) which, without the chimeric PrP 5 gene, might not be susceptible to infection with a human prion. is meant an immunoglobulin protein which is capable of binding an antigen.Antibodies as used herein are understood to include the entire antibody as well as any antibody fragments (for example) F (ab ") 2, Fab", Fab, Fv) capable of binding an epitope, antigen, antigenic fragment of interest. The antibodies of the invention are immunoreactive or immunospecific to and therefore specifically and selectively linked to a PrPSc protein or a soluble protein of the invention. Antibodies which are immunoreactive and immunospecific for native PrPSc and a protein of the invention are preferred. The antibodies, for PrPSo and soluble proteins of the invention are preferably immunospecific ie not substantially reactive with related materials. Although the term "antibody" covers all types of antibodies (eg, monoclonal), I antibodies are preferably produced using the I phage display methodology described herein. 125 By "purified antibody" is meant one which it is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such an antibody "binds preferentially" to a native PrPSc protein and a soluble protein of this invention (or an antigenic fragment thereof), i.e., substantially unrecognized and bound to other non-antigenically related molecules. A purified antibody of the invention is preferably immunoreactive with and immunospecific for both a prgthein of the invention and a PrPSc protein of a specific species and more preferably immunospecific for human PrPSo a related soluble protein of the invention. By "antigenic fragment" of a PrP protein is meant a portion of such a protein which is capable of binding to an antibody of the invention. By "specifically binding" is meant high capacity and / or high binding affinity of an antibody to a specific polypeptide, ie, epitope of a PrPs = protein. The antibody binding to its epitope in this specific polypeptide is preferably stronger than the binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest for example, binds more strongly to PrPSc than the denatured fragments of PrPc in such a way that when adjusting the binding conditions the antibody is j links almost exclusively to PrPs = and not to fragpjentos ! denatured of PrPc. The antibodies which are deployed I specifically to a polypeptide of interest may be able 1 to bind to other polypeptides at a weak level, still I 5 detectable (for example 10% or less of the link shown with the ! 'polypeptide of interest). Such a weak link, or background link, I is easily dissernible from the antibody binding specific to the compound or polypeptide of interest, for example, by the use of appropriate controls. In general, the antibodies of interest which bind to native PrPSc in situ with a binding affinity of 107 moles / 1 or more, preferably 10s / liters or More are said to bind specifically to PrPs. In general, | an antibody "with a binding affinity of 10 moles / liter or less is not useful since an antigen will not bind at a level I Il5 detectable using conventional methodology currently used.

| By "detectably labeled antibody", "anti PrP | detectably labeled "or" labeled anti-PrP fragment I "detectably" means an antibody (or fragment of I antibody which retains link specificity), which has 120 a detectable label attached. The detectable brand is linked I | normally by chemical conjugation, but where the brand is a polypeptide, it can be unit alternatively by genetic machining techniques. Methods for producing detectably labeled proteins are well known in the art. technique. The detectable marks can be selected from a variety of such marks known in the art, but 'normally they are radioisotopes, fluordforos, brands 1 paramagnetic, enzymes (eg, horseradish peroxidase), or ; other portions or compounds which either emit a I 5 detectable signal (eg, radioactivity, fluorescence, color) or emit a detectable signal after exposure j of the mark to its substrate. Well-known detectable label / substrate pairs (eg, horseradish / diaminobenzidine peroxidase, avidin / streptavidin, luciferase / luciferase IO), methods for labeling antibodies, and methods for using labeled antibodies in the art are well known (cf.

«Example, Harlo and Lane, Eds. . { Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The terms "treatment", "treating" and the like are used herein to mean generally obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of partially or completely curing a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease in a subject the which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibit the disease, that is, stop its development; or (c) cure the disease, that is, cause the regression of the disease. The invention is directed to treating patients with prion infections and is particularly directed towards treating humans infected with PrPSc, resulting in a disease of the central nervous system such as bovine spongiform encephalopathy, Creutzfeldt-akob disease, fatal familial disease or Gerstmann's disease. Straussler-Scheinker Abbreviations used here include: CNS for central nervous system, BSE for bovine spongiform encephalopathy, CJD for Creutzfeldt-Jakob disease, FFI for fatal familial insomnia, GSS for Gerstmann-Strassler-Scheinker disease, Hu for human HuPrP for a human prion protein, Mo for mouse, MoPrP for mouse prion protein, SHa for Syrian hamster, SHaPrP for Syrian hamster prion protein, Tg for transgenic; Tg (SHaPrP) for a transgenic mouse containing the PrP gene of a Syrian hamster; Tg (HuPrP) for transgenic mice containing the complete human PrP gep; 5 Tg (ShePrP) for transgenic mice containing the complete sheep PrP gene; Tg (BovPrP) for transgenic mice containing the PrP gene of whole cows; prpSc for the squeeze isoform of the prion protein; 10 PrPSc 106 for the squeeze isoform of prion protein that is soluble in mild detergents "consisting of residues 89-140 and 177-230; PrPc for the common contained cellular protein, normal isoform of the prion protein; 15 MoPrPSc for the scraper isoform of mouse prion protein; MHu2M for a chimeric mouse / human PrP gene wherein a region of the mouse PrP gene is replaced by a I I corresponding human sequence which differs from the 120 mouse PrP in 9 codons; j Tg mice (MHu2M) are transgenic mice of the invention, which include the chimeric MHu2M gene; j MHu2MPrPSc for the scraper isoform of the PrP gene of I j human / chimeric mouse; | 25 prpCJD for the CJD isoform of a PrP gene; Prnp ° / ° for ablation of both alleles of an endogenous prion protein gene, for example, the MoPrP gene; Tg (SHaPrP + / °) 8l / Prn-p0 / ° for a particular line (81) of transgenic mice expressing SHaPrP, + / 0 indicates heterozygote; Tg (HuPrP) / PrnP ° / ° for a hybrid mouse obtained by crossing a mouse with a human prion protein gene (HuPrP) with a mouse with both alleles of the altered endogenous prion protein gene; 10 Tg (MHu2M) / Prnp0 '° for a hybrid mouse obtained by crossing a mouse with a chimeric prion protein gene - (MHu2M) with a mouse with both alleles of the altered endogenous prion protein gene. FVB for a standard inbred strain of mice with I 15 frequency used in the production of transgenic mice, since the eggs of FVB mice are relatively large and tolerate the microinjection of exogenous DNA relatively well. GENERAL PERSPECTIVE Modified proteins are provided which retain abnormal properties associated with the disease of the native protein but which are made soluble by the modification. The modification can include all or any! of adding or replacing amino acids, eliminating amino acids or 'attaching another molecule to the native protein. A preferred embodiment of the invention is an amino acid sequence which is shorter than the native sequence and DNA codon segments that are expressed by a native protein are obtained in order to obtain a shorter, soluble protein which mimics the characteristics of a native insoluble protein - retains abnormal properties associated with disease . The soluble proteins of the invention are characterized by ": (1) include a structural modification of the full-length native protein; (2) having a greater degree of solubility than the native protein; (3) retain the biological characteristics basic protein abnormalities such as heart (a) that are not subject to enzymatic digestion and (b) that causes disease. The soluble proteins of the invention can be obtained by providing a DNA sequence encoding a ! native protein and systematically deleted codons. They are made and copies of shortened DNA versions are expressed for I provide the shortened proteins. Then the I proteins shortened for solubility. The proteins that retain their biological characteristics of the native protein. Other soluble proteins can be prepared modified from the invention and tested in a similar manner.

For example, a native sequence can be modified in such a way that particular codons are replaced with new codes expressing a different amino acid such as an amino acid. | polar (for example, glutamic acid, asparagine glutamine, and aspartic acid) which has hydrophilic properties. Can a large number of sequences are generated where different codons are replaced with new codons such as codons expressing a polar amino acid such as aspartic acid. The modified DNA sequences obtained are then expressed to obtain modified proteins. The modified proteins are tested for solubility. Proteins found to be soluble are further tested to confirm that they retain abnormal properties associated with disease. Soluble proteins that are greater than a native protein in a similar form can also be produced. The DNA sequence can be modified systematically by adding codpens within the sequence to either the end of the sequence. The modified DNA sequence is expressed to provide proteins that are larger than the native protein. The largest proteins are tested for solubility to confirm that they maintain abnormal properties associated with disease. In addition to shortening, lengthening, or modifying the amino acid sequence 0 by replacing one amino acid with another is capable of modifying the native protein by joining a new molcular entity. The aggregated molecular entity can be added by either linking to the termination of the native protein or by joining it anywhere along the chain.The typical 5 binding sites can include replacing an amino acid. with an amino acid substitute which includes an "R" group which is totally different from any "R" amino acid group found naturally and which increases the solubility of prptein. For example, the "R" group added may be a highly hydrophilic group. Finally, it is possible to modify the native proteins using combinations of all or some of the modifications described above. DETERMINATION OF SEQUENCE It is not part of the present invention to determine a nucleotide sequence which encodes a native, non-soluble protein. Such sequences are known, for example, the PrP gene sequences for several mam are described. { different feros within U.S. Patent No. 5,565,186 filed October 6, 1996 and the sequence or several beta-amyloid proteins related to Alzheimer's disease are described within U.S. Patent 5,387,746 filed February 7 from 1995. In addition to the nucleotide sequence information, the literature includes significant amounts of information on structural aspects of the protein expressed by the sequences. After obtaining a sequence of interest, the copies are made using conventional methodology, after making the copies, the copies are segregated in groups or reserves. For example, the copies can be divided into five more c, ten or ! more, one hundred or more, etc. groups or reservations of sequences. This separation is carried out in such a way that each group or reserve can be manipulated individually. Preferably "ten or more identical sequences are in each reservation. | 5 Once the reserves of identical sequences are obtained, the sequences within each of the reserves can be systematically altered. For example, the sequences ! within each reservation can be altered (i) eliminated one or I more codons (the cdons can be eliminated either from the extreme end of the protein or from the intermediate positions; (2) ; add additional codons (codons can be added to ! be the end or aggregates within the native sequence); (3) | add and delete codons. Depending on the information 'Known of the protein expressed by the genetic material, it is It is possible to eliminate or systematically add DNA segments which will eliminate or add particular structural characteristics of the expressed prctein. In this way it is possible to eliminate or add segments to propitiate segments of shortened or elongated DNA, which, when expressed, they will create different reserves of proteins, that is, i protein reserves where the protein reserves Different ones differ from each other because they lack or have a 'significant structural feature different. ! After expressing reservations. different from nucledtidos to obtain to obtain different reserves of I proteins, different reserves are tested individually Of proteins, first for solubility and after that for | biological activity. Those proteins which exhibit a significantly greater degree of solubility compared to the | 5 native protein are candidates for further testing. The I 1 determination if the modified protein is soluble may vary depending on the protein to be tested. For example, the | Protein can be tested in pure water, a normal solution similar to human body fluids or in an aqueous solution with a mild detergent. The solvent can be a solvent Watery and should be chosen in such a way that the native protein ! It is substantially insoluble in the chosen solvent so that i makes it possible to characterize the modified protein by its solubility within the solvent. In some cases it is desirable Í5 further ensure that the protein is soluble by subjecting the | proposed solution to a centrifuge at 100,000 g per I about an hour. If the protein does not precipitate and i remains in the supernatant, the protein is considered to be ! soluble. Those with experience in the technique will identify easily other solubility tests that are suitable for 'use with particular proteins. The soluble proteins that are identified are ! then tested by another trial such as the ability to ! the protein is digested with a specific enzyme, which does not digests the insoluble native protein. If you do not digest the protein, is then subjected to additional biological tests such as formulating the protein and inoculating an animal with the protein. The inoculated animal is observed to determine if the animal develops disease as a result of the inoculation. Transgenic animals that exhibit disease after inoculation with an infectious prion are described in a U.S. Patent No.No. 5, 565, 186 filed on October 15, 1996, incorporated herein and presented for reference to describe such transgenic animals and methods for testing compounds using such transgenic animals. INSOLUBLE PROTEINS The insoluble proteins of the present invention are modifications of insoluble native proteins that are associated with specific diseases. The invention is applicable to the modification of proteins that will still be discovered. However, the invention can now be applied directly to insoluble native proteins which are associated with particular diseases. The following is a non-limiting list of diseases with associated insoluble proteins. Disease Proteins insolub aa Disease of_Alzheimer Peptide APP, Aß, al- antiquimiotripsina, tan, component no aß Prion diseases, Creutzfeld's disease Jakob, scrapie and bovine spongiform encephalopathy PrPSc ALS SOD and neurofilament Pick disease Pick bodies Parkinson's disease Bodies of diabetes Type 11 Amiline Multiple myeloma - plasma cell disks Plasma IgL IgA polyneuropathy familial Transthyretin Medullary thyroid carcinoma Procalcitonin Chronic renal dysfunction ß2-microglobulin Heart dysfunction factor Congestive Atrial Natriuretic Sick Senile Cardiomyopathy and Systematic Transthyretin Chronic Inflammation Amyloid A serum Atherosclerosis ApoAl Familial amyloid Gelsolin It should be noted that the proteins listed above each include a number of variations or mutations that are proposed to be encompassed herein. Pathogenic mutations and polymorphisms occur in the PrP gene related to prion disease subsequently and the human, goat and bovine sequences are given in U.S. Patent No. 5,565,1865, filed October 15, 1996. MUTATIONS TABLE polymorph isirtos chivo 2 octarepeat Codon 129 Codon 171 5 or 6 insßrt Me / Val Arg / Glu octarepeats 4 octarepeat Codon 219 Codon 136 insert Glu / Lys Ala / Val 5 octarepeat insert 6 octarepeat insert 7 octarepeat insert 8 octarepeat insert 9 octarepeat insert Codon 102 Pro- Leu Codon 105 Pro- Leu Codon 117 Ala- Val Codon 145 Stop Codon 178 Asp- Asn Codon 180 Val- Ile Codon 198 Phe- Ser Codon 200 Glu- Lys Codon 210 Val- Ile Codon 217 Asn- Arg Codon 232 Met- Ala It should also be noted that such proteins often have two different three-dimensional conformations with the same amino acid sequence. One conformation is associated with disease characteristics and is generally insoluble and while the other conformation is not associated with disease characteristics and is soluble. The present invention attempts to modify the insoluble proteins in a form that renders them soluble but which allows them to retain substantially all of their characteristics associated with the disease. In this way when a soluble protein of the invention is used to inoculate an animal the animal will develop the disease. SYNTHETIC SOLUBLE PROTEINS The soluble proteins of the invention have at least two characteristics in common. First, are substantially more soluble than the native insoluble protein from which they are derived or on which they are based. Second, they continue to possess the abnormal properties of the native protein associated with the disease. The soluble proteins of the invention can be divided into a number of different categories. First, soluble proteins may have a lower number of amino acids than native insoluble proteins. Other soluble proteins of the invention have a higher number of amino acids than the native insoluble protein. Still others may have the same number of amino acids but , have different amino acids with some eliminated or added with respect to the native insoluble protein. In another modality the native protein is modified chemically including alternative amino acids or joining side chains! 5 chemistries which improve the solubility of the molecule. Finally, the soluble proteins of the invention can be dissolved. 'variations of all previous ones. The soluble proteins of the invention can be produced chemically They include minor, major or different amino acids compared to 0. 0 the insoluble native form or by the use of molecular genetics. Since it is often desirable to produce a greater number of different proteins initially, for testing, it is generally preferable to produce such proteins by the use of molecular genes. Proteins can be produced '15 include minor or greater numbers of amino acids_ or simply different amino acids compared to the native protein by the use of molecular genetics in a manner described below. Although it is not necessary to know the sequence is It is necessary to isolate the sequence which, when expressed, produces the insoluble native protein. Once this genetic material is isolated, it can be modified systematically. For example, a DNA sequence containing 100 codons can be copied and divided into 100 stocks different. A different codon can be eliminated in each 1 reservation. A variety of alternative systematic eliminations will be apparent to those with experience in the 1 technique For example, it can be eliminated every fifth codon or Codon groups can be deleted in a "termination ; 5 specific or internal areas of the sequence. In the same way it is possible to link the sequence of 'DNA at any desired point and add one or more codons | additional When codons are added, it is preferable to add Codons which encode polar amino acids and J10 particularly preferred to add codons encoding acidic acidic amino acids since they tend to provide I Increased solubility to the resulting protein. Those with | experience in the art will recognize that it is also possible 'simultaneously delete some codons and add other Il5 codons to reach the same number of original codoñes for 1 encode the native insoluble protein or arrive at a sequence I modified that includes a smaller or greater number of codons I compared to the sequence that encodes the native protein. Using the methodology such as that described in the? 20 United States Patent NO. 5,182,366 assigned to Huebner, I et al it is possible to generate thousands, ten thousand and even millions of different variations of a desired sequence in a relatively short period of time. I When desired sequences have been generated the sequences are then placed in an expression vector suitable using technology well known to those skilled in the art. The expression vector is then placed in a suitable host and expressed to obtain a large number of different amino acid sequences. The amino acid sequences are then tested using the concepts of the present invention in order to determine that the sequences are soluble. The soluble sequences are then further tested in order to determine whether they continue to retain the abnormal properties associated with diseases they possess. Í0 properties of the native insoluble protein. Those proteins ! which are both soluble and possess the abnormal properties associated with the disease are proteins of the invention. i Chemical synthesis methodology can be used to produce additional variants in which variants can to be tested also for solubility and for activity 1 biological with respect to maintaining abnormal properties I associated with disease that are possessed by the protein Insoluble native. Can be applied the "methodologies of I chemical synthesis to produce proteins that include uncoded amino acids. In addition, the amino acid sequence It can be chemically synthesized to include optical isomers of naturally occurring amino acids I that is, the D forms. Several non-amino acids are described Coded and methods to produce millions of variations of such within U.S. Pat. No. 5,420,246, ceded to Rutter, et al on May 30, 1995. Chemical methodologies make it possible to produce a large number of Different variations of amino acids. In such example is described within PCT Publication WO94 / 06451 assigned to Zuckerman, et al., Published March 31, 1994. When such methodologies are performed for the purposes of the present invention, the "R" groups of the amino acids or amino acid substitutes are generally polar groups that provide ! 1 a hydrophilic property to the molecule as a whole so 0 provide the solubility characteristics of the molecule. Although chemical synthesis methodologies can be used to produce sclubles mclecules those molecules I may be less useful in terms of generating : antibodies The antibodies generated using such molecules can have affinity to sites that do not exist in the native inscrutable molecule. Thus, for purposes of the present invention, it is preferred to use the amino acid sequences " 1 short ones "that are soluble in order to generate antibodies that bind not only to the shortest soluble sequence but to the entire full-length native insoluble sequence." PRODUCE ANTIBODIES "When soluble proteins have been created and tested of the invention for biological activity, those proteins are extremely useful for generating antibodies that bind to the native protein as well as the soluble protein. Such antibodies can be produced by first inoculating a host animal with a native insoluble protein I (or alternatively, a soluble protein in the invention). The host animal can be any animal, and is preferably a host animal of the type defined in the present invention such as a mouse, rat, guinea pig, or hamster and is more Preferably a mouse. The host animal is inoculated with proteins that are endogenous to a different species that is preferably a genetically diverse species as defined herein. For example, a transgenic mouse is inoculated with human PrPSc. The inoculated host animal I 1 will then generate antibodies. It is preferable to inoculate an animal which has the endogenous gene that expresses the protein ! ablative For example, a mouse that has its endogenous PrP gene 5 ablative mouse that is described in the Patent of the States United States No. 5,565,185. A generalized method for «Ablative an endogenous gene in the Patent of the States 1 U.S. No. 5,464,764, assigned on November 7, 1995, to i Capecchi et al. 20 After allowing the generation of antibodies the | mouse is sacrificed and cells are removed from the bone marrow and ! of the spleen. The cells are lysed, the RNA is extracted and I transcribed in reverse form to cDNA. Then the I heavy and light chains of antibodies (or parts of the same) by CPR. A cDNA library can be used j amplified as it is or after manipulation to create a range of variants and therefore the size is incare ! from the library. A biblicteca of exhibition of phage IgG by inserting the amplified DNA encoding a chain Heavy IgG and the amplified cDNA encoding a light chain in a phage display vector (e.g., a pComb3 vector) such that a vector contains a cDNA insertWhich encodes a heavy chain fragment in a first cassette JLO of vector expression, and a cDNA insert encoding a I j fragment of light chain in a second cassette of expression i of the vector.; The bound vectors are then packaged by M13 phage [filamentous using methods known in the art. It's used Then the library is packed to infect a culture of E. coli, in order to amplify the number of phage particles.

'After bacterial cell lysis, isolates are isolated ', phage particles and are used in an expansion procedure. The proteins of the present invention are invaluable in the expansion procedure. Since the proteins are soluble, they can be more easily contacted with the phage particles to determine which -hares are expressing the antibodies that bind to proteins of I the invention. The proteins of the invention can be labeled in order to detect more easily when the link occurs.

I Those phages that bind proteins of the invention are then isolated and cloned. The genetic material can be cellocated in cells to produce antibodies. The cells can be fused with appropriate melanoma cells with the! 5 to create hybridomas for the production of monoclonal antibodies I. ! EXAMPLES 1 The following examples are given to provide a Those skilled in the art with a complete description of how to make soluble proteins and perform the methodology for finding such proteins, and are not proposed to limit the scope of what is taken into account as the invention. Efforts have been made to ensure accuracy I with respect to numbers used (for example, quantities, '15 temperature, etc.) but some must be taken into account 1 experimental errors and deviations. Unless indicated I 1 otherwise, the parts are parts by weight, the molecular weight is I average molecular weight; the temperature is in degrees Celsius; and the pressure is at or near atmospheric. '20 EXAMPLE 1 j ELIMINATION OF PrP 'SEGMENTS In a specific modality, they are eliminated I systematically segments the Prp molecule. Specifically, regions of putative secondary structure in PrPc are deleted, Z. Huang, et al., Proc. Nati Acad. Sel. USA 91, 7139-7143 (1994). The elimination of each of the four putative helices prevents the formation of PrPSo. However, eliminating a 36 residue circuit between the two and three blades is not harmful. A resulting PrPSc molecule is designated PrPSc 106 since it contains 106 amino acids after cleavage of an N-terminal signal peptide. While PrPSc exhibits resistance to digestion by proteinase K, it is soluble in 0.5% sarcosil. The discovery of PrPsp 106 greatly facilitates the structural studies of PrPSc, as well as | 10 also investigations of the mechanism of formation of PrPSc and l the production of specific antibodies to PrPSc. I During the conversion of PrPc into PrPSc PrP suffers a 'change conformations deep, K.M. Pan, et al .. Pro. Nati.

, Acad. Sci. USA 90, 10962-10966 (1993). This transition I 15 structural is accompanied by the acquisition of insolubility I in non-denaturing detergents and resistance to digestion 'by proteinase K, S.B. Prusiner, et al., Biochemistry 21, J 6942-6950 (1982); R. Meyer, et al., Proc. Nati Acad. Sci.

USA 83, 2310-2314 (1986). The structural studies of PrPsc have been limited by the insolubility of the molecule. S.B.

I I Prusiner, et al., Cell. 35, 349-358 (1983); B.W. Caughey, Et I 1 al., Biochemisry 30, 7672-7680 (1991); M. Gasset, et al-, Proc.

'Nati. Acad. Sci. USA 90, 1-5 (1993); J. Safar, et al., J. Biol.

Chem. 268, 20276-20284 (1993). However, the synthetic and recombinant fragrances of PrP to PrPSo analogues have been more suited to structural research, H. Zhang, et al., J. Mol. Biol. 250, 514-526 (1995); I. Mehlhorn, et al., Biochemistry 35, 5528-5537 (1996); R. Riek, et al., Nature 382, 180-182 (1996). The studies are limited not only by insolubility but by difficulties in achieving high level of expression of non-degraded recombinant PrP. I. Mehlhorn, et al., Biochemistry 35, 5528-5537 (1996); M. Scott, et al., Protein Engineering 2, 69-76 (1988). EXAMPLE 2 10 CLONING OF PrP Molecular cloning of a cDNA encoding PrP of the pellet is performed to study the structural characteristics of PrP of mammal by molecular modeling. Four regions of putative secondary structure are identified 0. 5 using various structure prediction algorithms, Z. Huang, et al., Proc. Nati Acad. Sci. USA 91, 7139-7143 (1994); M. Gasset, et al., Proc. Nati Acad. Sci. USA 89 10940-10944 I (1992). Although this is in disagreement with whether certain regions adopt structures - helix or ß leaf, all an? } lysis and 20 predict that these regions can adopt structures high schools. Accordingly, the peptides are produced Synthetics corresponding to each of the four regions I designated Hl, H2, H3 and H. In aqueous shock absorbers Hl, H3, and I H4 the structures unexpectedly adopt the structures of o ß leaf, M. Gasset, et al., Proc. Nati Acad. Sci. USA 89 10940-j i 10944 (1992). EXAMPLE 3 STRUCTURAL ANALYSIS Mix Hl in a shaping of ß sheet, with H2 in a coil acts to convert H2 to or ß leaf,, J. Nguyen, et al., Biochemistry 34, 4186-4192 (1995). A peptide larger than 56 residues containing Hl and H2 adopts the a-helix conformation in aqueous buffers exhibit chemical changes indicating i an a-helix in the Hl, H. Zhang, et al. , J. Mol. Biol.

I 10 250, 514-526 (1995). The secondary structure as determined 'by NMR in the H2 region is less clear. The H3 and H4 regions are identified in the NMR studies of a C fragment ! terminal 111 residues, R.Riek, et al., Nature 382, 180-182 I (1996). Those studies also identify an a-helix in 0. 5 the circuit between H2 and H3 as well as ß-short sheet that i consists of residues 128-131 and 161-164 that forms chains ! antiparallel. j j Based on the molecular model of PrPc as well as To IPS data from CD and FTIR studies, tests are carried out I B0 systematic model in terms of the capacity that mutated PrPSc molecules are converted into PrPSc. The studies are done in both cultured neuroblastoma cells (N2a) And transgenic mouse (Tg). As a result, a I map of the regions of secondary structure putatiya in E5 terms of the requirement for each region to support the formation of PrPSo (Figure 1). I EXAMPLE 4; EPITOPE MARKS - ANTIBODIES I Studies are carried out on N2a cells infected with i 5 scraper (ScN2a) using a molecule marked with epitope MHM2 PrPc which has been previously shown to be converted I in MHM PrPs ° in ScN2 cells. A. Taraboulos, et al., Proc.

I Nati. Acad. Sci. USA 87, 8262-8266 (1990) f M. Rogers, et.

Immunol. 147, 3568-3574 (1991); M. R. Scott, et al-, Protein '10 Sci. 1, 986-997 (1992). All PrP constructions are cloned I, to be expressed in ScN2a cells in the vector pSPOX-llpeo and The cells are transfected temporarily, M.R. Scott, et al., Protein Sci. 1, 986-997 (1992). | PrP MHM2 marked with epitope has two Met residues 0. 5 in positions 108 and 111 that are in Sha and Prp. HE Recognizes this epitope by a-PrP 3F4 mAb, Rogers, et al., And Immunol. 147, 3568-3574 (1991); R.J. Kascsak, et al., J. Virol. 1 i 61, 3688-3693 (1987). Since PrPc is easily converted by i MHM2 into PrPSc in ScN2a cells, it can be used to to evaluate the effect of selective deletions of specific i domain- within the PrP molecule (Figure 1). ! EXAMPLE 5 «EXPRESSION IN CELLS ScN2a MHM2 is used in which the N-terminal region that! 25 consists of the waste 23-88 has been removed as a Starting material. This construct supports formation of PrPSc in ScN2a cells as evidenced by the production of a reactive protein α-Prp 3F4 mAb which is resistant to proteinase digestion, M. Rogers, et al., Proc. Natl._Acad. Sci. USA 90, 3182-3186 (1993). When residues 33-80 of MoPrp are removed, it is shown that the resulting construction leads PrnP0 / 0 mice to be susceptible to scraper prions.

Mo, M. Fischer, et al., EMBO. J., 15.! Three regions of secondary structure are eliminated putative designated H2, H3 or H4. In separate constructions I the H2, H3, and H4 regions are eliminated from the MHM construction I 2 PrP terminally truncated in N in which they have been I | eliminated the residues 23-88. Each of these PrP eliminated ; it is then expressed in ScN2a cells. The H2 regions (residues 122-140), H3 (residues 177-200) and H4 (residues 201-? 217) are removed and each construct expressed in cells $ cN2a ! is detected by immunostaining with a-PrP 3F4 mAb. The H3 elimination which is not large contains both consensus sites for glycosylation linked to Asn, C. Locht, et al., Proc. Nati Acad. Sci. USA 83, 6372-6276 (1986) migra corao a j band of Mr -19 k Da. Two other constructions of the circuit are also expressed between H2 and H3 containing 36 ! residues (141-176) and the other with a point mutation in the '• residue 178 where Ala is replaced by Cis to avoid formation of a disulfide bond between this residue and Cis 213.

EXAMPLE 6- RESISTANCE TO PROTEINASE k The elimination of the H2, H3, and H4 regions as well as I also the mutagenesis of Cis 178 prevents the formation of PrPSc I I 5 as judged by resistance to digestion with proteinase K (20 μg / ml, 30 min., 37 ° C). The results show that MHM2 is I converted to a protease resistant isoform as the N-terminally deleted molecule without residues 23-88. The elimination of the circuit between H2 and H3 that contains 36 residues (141-176) does not prevent the conversion of the molecule into a protease-resistant isoform. The acquisition of protease resistance during training ! of JPrPSc seems to occur within the domains similar to caveloas (CLD) and PrPc is objectivated to CLD by its GPI anchor. [L5 A. Taraboulos, et al., J. Cell Biol. 129, 121-132 (1995). HE I expose the cells expressing the construction with the I elimination of circuit to PIPLC. The results show that | Virtually all MHM2 digestible by protease (from 23-88, I 141-176), designated PrP 106 is released by the enzyme. N.

Stahl, et al, Cell 51, 229-240 (1987). This indicates that PrP 106 I transits from ER to the cell surface where it is linked per I GPI anchor as full length PrPc. EXAMPLE 7 | REGION ELIMINATED (STE) 25 An analysis of waste disposal is carried out i containing HL 108-121 creating an 1138M mutation in order to produce epitope ShaPrP detectable by a-PrP 13 * 5 mAb, M.

I Rogers, et al., J. Immunol. 147, 3568-3574 (1991; RA Barry,! Et al., J. Infect. Dis. 154, 518-521 (1986).) As the elimination of H2-H4, elimination of Hl prevents I-formation of PrPSc. After this, a-PrP 13A5 mAbs is used to study the elimination of the arrest transfer effect (STE) and region composed of residues 95-107 adjacent to Hl that have been implicated in the control of PrP translocation. 10 synthesized in cell-free systems with microsomal membranes from dog pancreas CS Yost, et al-, Nature 1 343, 669-672 (1990) As eliminations of H1-H4, the elimination of the STE region also avoids the formation of | PrPSc. 15 Based on the previous results, changes in M r values are examined after limited digestion with protease K for PrPsc 106. In the absence of proteinase 'K, both isoforms MHM PrPc and PrPS encoded by the I transfected constructs are immunostained with a-PrP 3F4? 20 mAbs. The broad range of Mr values is presumably due to the glycosylation linked to aberrant Asn. After the limited protedysis the Mr values are decreased and the range of molecular Mr values decreases. Based on the shift in the Mr values, it seems likely that they could have eliminated amino acid residues from the termination.

N of PrPSc 106 during limited proteolysis. Microsequencing can provide resolution of this tissue. EXAMPLE 8 5 SOLUBILITY - I Insolubility in non-denaturing detergents 1 has been an invariant feature of protease resistant PrPSc. R.K. Meyer, et al., Proc. Nati Acad. Sci. USA 83, 2310-2314 (1986); S.B. Prusiner et al., Cell. 35, 349-358 '10 (1983). Knowing this, ScN2a cells expressing PrP 106"are used in increased concentrations of N-lauroyl sarcosine (sarcosil) .The suspensions are taken to a centrifuge at 100,000 x g for 1 hour at 20 ° C. ! 0.5% concentration of sarcosil or more, all the PrPSc 1-06 is found in the supernatant. When probing again I I blot to detect MoPrpSc with rabbit polyclonal a-PrP antiserum R073, all immunostaining is found in the granule fraction and nothing in the supernatant. 1 EXAMPLE 9- I 20 STRUCTURAL ANALYSIS 1 The results obtained show that "all four putative helical regions identified by ! molecular modeling and the STE region discovered in the studies '- free cell translation for PrPSo training. The results also show that the disulfide bond between the Cis residues 178 and 213 entered into both PrP ° and PrPSo, E. Turk, et al, J. Biochem. 176, 21-30 (1988) is essential for the formation of PrPSc. Although alteration of this disulfide by the C178A mutation decreases the conversion of this mutated Prpc j 5 into PrPS the treatment of highly concentrated fractions < for doctor blade infectivity with either 2% ß-mercaptoethanol 1 or 100 mM of dithietreitol does not decrease prion titers | as measured by bioassays in Syrian hamsters, S.B. Prusiner, et al., Biochemistry 19, 4883-4891 (1980). 10 When the recombinant PrP (90-231) is deduced, which corresponds to the residues found in PrP 27-30 (the nucleus Protease resistance of PrPS) after purification under denaturing conditions, this adopts a structure with a high content of a-helix similar to PrPc of mammal after the formation of the disulfide bond. I.

Mehlhorn, et al., Biochemistry 35, 5528-5537 (1996). It is believed ! that the disulfide stabilizes the terminal α-helices in C ie, H3 and H4. Z Huang, et al., Proc. Nati Acad. Sci. USA 92, 7139-7143 (1994); Z. Huang, et al., Folding & Design 1, 13-20 (1996). This is supported by NMR structural studies of a C-terminal fragment of recombinant PrP (121-231) i expressed in E. cpli. R. Riek, et al., Nature 382, 180-182 S (1996). It is unknown if rPrP (121-231) which lacks the I 99 N-terminal amino acids as well as CHO bound to Asn and the GPI anchor reflects exactly the structure of the region C terminal of PrP0. however, the data of the de_rPrP study (121-231) demonstrate a-helices that are predicted for the H3 and H regions. EXAMPLE 10 CHARACTERISTICS OF PrP 106 PrP 106 is converted, "" which lacks residues 23-88 to a molecule similar to PrPSc *. This supports the position that the region cuts from ß leaf formed by two antiparallel ß chains consisting of residues 128-131 and 161-164 IjO are likely to be a nest in which the PrPSc starts. R.

Riek, et al., Nature 382, 180-182 (1996). Additionally, I do not know I i also requires the third a-helix (144-154) found in j rPrP (121-231) within the putative circuit for the Acquisition of resistance to limited proteolysis. 15 The results shown here are the I I first to decouple protease resistance and insolubility of PrPs. Such results support the position that the ! Aggregation which should contribute to the insolubility of PrPSc 1 purifca is not required for protease resistance. 20 Recent studies of cell-free mixtures of the "Linkage of partially denatured PrP ° to PrPSc have argued that the protease resistance exhibited by radiolabelled PrPc i" is inseparable from its binding to PrPSe aggregates.

! GIVES. Kocisko, et al., Nature 370, 471-474 (1994); B. Caughey, 2 | 5 et al., Jr. Chem. Biol. 2, 807-817 (1995). However, the Results of the present demonstrate that insolubility and protease resistance are separable. These results coincide with recent studies in which PrP 27-30 in rod-shaped aggregates is dispersed in the liposomes and remains resistant to proteolytic digestion. R. Gabizon, et al., J. Biol Chem. 263, 4950-4955 (1988). The PrPSc 106 shown is less soluble than full-length PrPc but substantially more soluble than full-length PrPSc. This suggests that the interaction between the 0 PrPso 106 molecules (or protease resistant units composed of PrPs = 106 molecules) is altered by detergents more easily than the interaction between full length PrPS molecules. When using Tg mice with an ablative background with PrP for 5 studying the formation of prPSc 106 it will be confirmed that _Prpso j supports prion infectivity. Additional study to determine if PrPSc supports amyloidogenicity in vivo as well as in vitro can demonstrate that it binds decoupling from; infectivity and amyloidogenicity to the solubility of the 20 molecules. ! The unique intermediate solubility of PrPSc will simplify Its purification. First, PrPs will be separated from PrP 106 Using weak detergents, then separated from other Prpsc molecules , using stronger detergents. Skip the procedure I 25 protease digestion of purification protocols will be advantageous for structural studies which can be altered by the presence of proteases. j EXAMPLE 11 j FORMATION OF ANTIBODY I The ability of ScN2a cells to form a The protease resistant but soluble Prp c molecule will simplify the isolation of PrPSc-specific antibodies by display of fgos. Specifically, find an antibody PrPsc will be greatly facilitated by expanding and evaluating against 1 a soluble compound such as a PrPsc 106 micleable solvable.

A.A. Williamson, et al., Proc. Nati Acad. Sci. "USA 93 7279-7282 (1996) Determine the tertiary structure of PrPsc should I be substantially advanced by availability of PrPSa 106 as it should be deified from the mechanism by which it becomes PrPc in PrPsc. The formation of a soluble form of PrPsc in ScN2a cells can also facilitate the development of an effective pharmacotherapeutic drug for prion diseases. F. E.

Cohen, et al., Science 264, 530-531 (1994). In addition, they can be "tested soluble proteins of the invention such as PrP 106 and variations thereof, for their effect in decreasing the disease progression associated with PrPsc. Since the prion diseases are unprecedented in biology and medicine, it is likely that the design of a drug to treat these disorders will require a detailed understanding of the structural transition that PrP undergoes as soon as PrPSc is formed. 5 With the possibility that bovine prions have been I transmitted to people in Brittany and France causing vCJD G.

Chazot, et al., Lancet 347, 1181 (1996); R.G. Will, et al., Lancet 347, 921-925 (1996), the development of an effective therapy for prion diseases has acquired a giant importance. When using soluble proteins of the invention to generate antibodies it is generally preferable to use soluble proteins which include a smaller number of amino acids than the native insoluble protein. By using smaller proteins it is possible to avoid the generation of antibodies which bind to amino acids which have been added and which antibodies may be irrelevant in terms of having binding affinity to the native insoluble protein. The present invention is shown and described herein in what is considered to be the most practical and preferred modalities. It is recognized, however, that modifications can be made, which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art after reading these descriptions.

Claims (10)

CLAIMS! 1 1. A soluble form of a protein insoluble in its native form, produced by a process selected from the group 5 which consists of: (a) removing amino acids from a sequence of native amino acids; (b) adding amino acids to a sequence of native amino acids; 10 (c) linking a hydrophilic molecular moiety to a native amino acid sequence; and 1 (d) a combination of any of (a) - (c), wherein the Soluble protein produced retains properties of the | Insoluble native protein associated with disease

1.5 2. The soluble protein according to the ! claim 1, characterized in that the protein is produced 'by a process comprising the steps of: I obtaining a DNA sequence that encodes a naturally occurring protein which has a 20 insoluble conformation; make copies of the DNA sequence; eliminate a plurality of different codons from a | plurality of DNA sequences encoding native insoluble protein j to provide a plurality of sequences

2.5 different DNAs which differ from each other by the deleted codons; expressing the different DNA sequences to obtain a plurality of different proteins; test the proteins for solubility and biological activity; and isolating a protein with a higher solubility and substantially the same biological activity as the native protein with respect to the properties associated with disease wherein the protein that is found naturally in an insoluble form or conformation is selected from the group consisting of 'a PrPSc protein; a ßamiloid protein and a body Le and. i 3. The protein according to the claim 1, characterized in that the native, insoluble form of the protein causes a disease of the central nervous system 15 in a mammal where the disease is a disease selected from the group consisting of Alzheimer's Disease, i 1 Prion diseases. Creutzfeld Jakob Disease, I Scrapie and Bovine Spongiform Encephalopathy, ALS, Pick I Disease, Parkinson's Disease, Type 11 Diabetes, Multiple Myeloma - Plasma Cell Discrecies, Polyneuropathy

! ! Familial amyloidotica, Medullary thyroid carcinoma, Chronic renal dysfunction, Congestive heart dysfunction, Senile and systematic cardiac amyloidosis, Critical inflammation, Atherosclerosis, Familial amyloid. 5 4. The soluble protein according to claim 1, characterized in that the protein is enhanced by a process comprising the steps of: determining a DNA sequence that encodes a naturally occurring protein with an insoluble conformation; make cepias of the DNA sequence; adding a plurality of different codons to a plurality of DNA sequences encoding the native insoluble protein to provide a plurality of different DNA sequences or which differ from each other by the aggregated codons; expressing the different DNA sequences to obtain a plurality of different proteins; prpbar the proteins for solubility and activity 3-5 biological; and 1 isolating a protein with greater solubility and substantially the same biological activity as the native protein with respect to properties associated with disease; where the protein found naturally

I with an insoluble conformation is selected from the group consisting of APP peptide, Aß, al-antiquimiotripsin, non-Aß component, PrPSo, S0D and neurofilament, pick-up, amylin, 'chain IgL, transthyretin, procalcitonin, ß-microglobulin, I I atrial natriuretic factor, transtrietin, amyloid A serum, 125 ApoAl, Gelsolin.

5. A modified PrPSc protein characterized by a capacity to be digested by protease and has twice the solubility or more as the native PrPsc.

6. The modified PrPSc protein according to claim 5, characterized in that it also has the solubility of native PrPsc cince vecee and causes decrease in a mammal and still further characterized by the lack of a circuit structure in which the structure i of circuit is present in the native PrPsc and contains 10 generally about 36 amino acid residues.

7. The modified PrPSc protein according to claim 5, characterized in that the protein is PrPsc j 106.

8. An antibody which selectively binds to the soluble protein according to claim 1,

9. A method to test a sample for the ! presence of an insoluble native protein associated with disease, characterized in that it comprises: I] Contacting the sample with the antibody of the 20 claim 8; Y ! Determine the amount of binding between the antibody and the sample to thereby determine whether the native protein Insoluble associated with the disease is present in the sample.

10. A method for evaluating drugs, characterized because it comprises: Solubilizing the soluble protein according to claim 1 to create a solution; Put a drug in contact with the solution; and Determine the effect of the drug on properties of the soluble protein associated with disease.