JPH10506271A - Methods for detecting abnormally folded proteins - Google Patents

Abstract

(57) Abstract: The present invention provides a new method for detecting abnormally folded proteins; a method for measuring the amount of abnormally folded proteins produced; and a method for detecting compounds that affect abnormally folded proteins. About.

Description

DETAILED DESCRIPTION OF THE INVENTION Methods for detecting abnormally folded proteins   The present invention provides a new method for detecting malfolded proteins; Method for measuring the amount of abnormally folded protein produced; and abnormally folded protein The present invention relates to a method for detecting a compound that affects tamping.   When the abnormally folded protein accumulates in the cell, a gene encoding a specific protein Transcription of the gene is induced in the nucleus. This “unfolded protein response” It was first studied in animal cells. Here, Bip / GRP78, GRP94, PDI / ERp59 and ERp 72 were induced by a variety of different treatments, leading to an increase in abnormally folded proteins. It is. Response to abnormally folded protein accumulation is aided by specific enhancer factors Is regulated at the transcription level.   A similar response to abnormally folded protein accumulation isSaccharomyces cerevisia D (Saccharomyces Cerevisiae) Has also been observed. Here, (KAR2 Bip, PDI, Euglp and peptidyl-prolyl cis-tolan Expression of a similar set of genes including suisomerase is induced. KAR2 Promo Using deletion analysis together with the unfold protein response factor (UPR ) Have been identified. This UPR activates transcription in response to abnormally folded protein accumulation. Necessary and sufficient for activation.   The biological function of a protein is governed by a particular three-dimensional structure. Abnormal The newly folded or aggregated protein is a necessary conformation required for the activity of the protein. Fail to get the animation. Normally, abnormally folded proteins in cells Accumulation of Alzheimer's disease (AD) and Creutzfeldt-Jakob disease and Gerzmann-ost Directly or indirectly to such human prion-related diseases. Abnormally folded protein Another example of the effect of tumor suppressors is the tumor suppressor protein, a negative regulator of cell proliferation Quality p53 (Donehower & Bradley, Biochim. Biorhys. Acta (1993), 1155, 81). ~ 205). Most human tumors express a variant p53 protein. Therefore, the mutant p53 protein Is believed to be abnormally folded or at least partially denatured.   Alzheimer's disease is pathologically linked to, for example, the appearance of cerebrovascular amyloid sediment doing. The major proteins found in these sediments are mainly cross-β-con 39-42 amino acids called β-amyloid peptide in the form of home Is a peptide. From cDNA analysis, β-amyloid peptide was found to be It has been shown that amyloid precursor protein can be obtained. APP has many details Produced in a cell type and usually does not aggregate. In contrast, the synthesis of β-amyloid Adult molecules have been shown to aggregate (Barrou et al., J. Mol. Biol. (1992)). , 225, 1075-1093). In cells, the first protein that abnormally folds is the most Aggregates eventually, but the aggregated protein is not thought to be unnecessarily folded ing.   In this familiar finding, because of their effect on abnormal protein folding, For example, because of their ability to minimize the production of certain abnormally folded proteins To screen for compounds, use the negative effects of the abnormally folded protein produced To reduce or offset the effects that cause the protein to fold abnormally. It is very valuable to have tests that allow a quick and easy way to There will be. Summary of the Invention   Surprisingly, the effect of the compound on the abnormal folding of the protein was White matter acts on a signal sequence that directs the transport of this protein through the endoplasmic reticulum (ER) If one or more UPR factors operatively bind to the reporter factor If monitored, it can be monitored by easily measurable reporter gene products. Was found. The method of the present invention relates to the abnormal aggregation of proteins associated with abnormal folding. It also provides an easy way to determine if they are linked. For example, β-amyloid In the case of peptides, surprisingly, aggregation in vivo and in vitro leads to abnormal folding. It has been found that it can be monitored in vivo using the system of the invention. ing. In addition, as estimated in vitro (shortened, inverted, or The ratio of aggregation of different β-amyloid peptides (from different organisms) Closely related to the percentage of abnormal folding measured in the system. Therefore, abnormal folding It is also possible to monitor the effect of the mutation on the fold ratio and its associated effects. It is possible. Detailed description of the invention   The present invention relates to a host transformed with at least the first and second expression cassettes. What   -One or more folders wherein said first expression cassette is operatively linked to a reporter element; Including an unfolded protein response factor (UPR);   -The second expression cassette is monitored for abnormal folding in the signal sequence. Operably linked to the DNA encoding the protein to be processed and to the terminator Including a designated promoter; and   If the first and second expression cassettes are not native in the host, Protein to be monitored in said second expression cassette Quality is from prion, p53, β-amyloid peptide and their functional derivatives A host selected from the group consisting of:   Host   Suitable hosts according to the present invention are "amphos," such as hosts that secrete proteins through the ER. Any host capable of producing an "unfolded protein response" obtain. Examples for suitable hosts are plants, insects, mammals, fungi or animal cells. is there. Preferred are fungal cells, more preferably yeast cells, and And most preferablySaccharomyces cerevisi ae ).   A suitable yeast strain according to the present invention is a yeast strain containing an exogenous 2 micron plasmid.. C S. cerevisiae ) Or treated with said exogenous 2 micron plasmid These strains have been identified (see EP-A-340170).   Hosts are commonly used in genetic engineering and are described, for example, in Sanbrook et al., Molecular cl. oning; A laborotorymanual, 2^nd Edn. Required by the means described in 1989 Transformed with the first and second expression cassettes.   Expression cassette   Unhalted protein responsive element as used in the first expression cassette (UPR) is a measure of the DNA involved in transcription initiation when abnormally folded proteins accumulate. It is a fragment. UPR factors include, for example, BiP, FKB2, BiP / GRP78, GRP94, PDI / ER p59 and ERp72 (Shamu et al., Trends in Cell Biol. (1994), 4, 56-60) Such as those included in response to the accumulation of abnormally folded proteins It can be isolated from the start region of the protein.   Preferred UPRs are isolated from BiP, more preferably as shown in SEQ ID NO; Or a functional equivalent thereof. Functional equivalent collapsed At one or both ends that do not interfere with its activity in response to accumulation of an uncommon protein For some substitutions of the restriction sites, or for example for the substitution of 1 to 5 nucleotides Has the meaning of the DNA sequence obtained from SEQ ID NO: 1. Stronger response to accumulation For example, a UPR may be present in one or more copies, for example, 2-5 copies.   In a preferred first expression cassette of the present invention, the reporter element comprises It is operably linked to DNA transcribed under the control of a motor and to a terminator. Includes promoters.   The promoter in this reporter element can be from almost any source. obtain. For example, a β-galactosidase or luciferase promoter, or Tightly regulated promoters such as CYC1, Gal1 / GA119, PHO5 or KAR2 promoters Promoter or promoter naturally adjacent to the DNA. You. In a preferred embodiment of the invention, the promoter is a CYC1 and KAR2 promoter. Selected from the group consisting of   Suitable DNA transcribed under the control of this promoter is either during transcription or translation or It usually causes an effect that can easily be measured later. Preferred is, for example, produced Can be easily measured by measuring the amount of protein mRNA or DNA; or Effects that can be determined, such as cell growth, enzymatic reactions or color-induced transcription or translation It is a translation. Thus, the transcribed DNA is, for example, Produced in a quantity related to the quantity, e.g., in a host selected under the conditions applied. Encodes a protein that is not produced elsewhere. An example of a suitable protein is EastCUP1Gene, β-galactosida Metallothionein encoded by luciferase or luciferase. preferable Are luciferase and β-galactosidase.   A suitable terminator for this first expression cassette is Appropriate signals will usually also be included. This terminator naturally binds to the transcribed DNA. Can be combined, orPHO5, Α-factor orSUC2A different source like a terminator Can be introduced.   This first expression cassette comprises, for example, a signal peptide as defined below. May further comprise a DNA sequence encoding   The promoter for the second expression cassette according to the present invention is It can be almost any homologous or heterologous promoter. For example, its abnormal A protein originally bound to the DNA encoding the protein whose folding is to be monitored. Or it is common in genetic engineering of hosts. May be used as a promoter. Promoters are CUP1, GAPDH, GAPFL, GAL (1/10), PYK, TPI, ADH, PRC1, and PGK promoters Cannot be induced. Preferred is the GAPFL promoter or a functional derivative thereof. is there.   If an inducible promoter is used, the host can be grown under optimal growth conditions. , The expression of the protein whose abnormal folding is to be monitored takes the required time, Can be introduced.   The signal sequence is usually selected according to the host used for the test. Appropriate The signal sequence may be any gene encoding a polypeptide secreted from the source. Can be obtained from Examples include SUC2, CPY, α-factor, KEX1, PHO5 and Glucoa This is a myrase signal sequence.   The proteins whose abnormal folding should be monitored are prions, p 53, β-amyloid peptide or a functional derivative thereof. Preferred of the present invention In some embodiments, the protein to be monitored is a β-amyloid peptide or Include functional derivatives thereof. The term functional derivative refers to its abnormal folding Small transformations with similar or identical properties, e.g., substitutions of up to 10 amino acids, Refers to a peptide obtained from the original unfolded peptide by addition or deletion.   The terminator used for the second expression cassette has a abnormal folding. A terminator naturally adjacent to the DNA encoding the protein to be It may be another terminator described above.   A promoter, a DNA sequence encoding a signal peptide, and a polypeptide And the DNA sequence containing the transcription termination signal are operatively linked to each other. . That is, they are arranged in such a way that their normal function is maintained. That arrangement The columns indicate that the promoter is responsible for proper expression of the signal sequence-polypeptide gene complex. Transcription termination signal provides for accurate termination of transcription and polyadenylation And the last codon of the signal sequence is the first codon of the gene for the polypeptide. A polypeptide gene in its exact reading frame in such a way as to bind directly to the don It is like being combined. The promoter is the main mRNA initiation and promoter It is preferably linked to a signal sequence between the ATG naturally linked to the gene. The signal sequence has its own ATG for transcription initiation. Combining these sequences Are synthetic oligodeoxynucleotides having, for example, an endonuclease recognition sequence. It can be provided by means of a tidlinker.   An expression cassette according to the invention can be inserted into the host genome or It can be inserted into the form of a stable plasmid, such as a plasmid. Expression cassette One or both of the polypeptide expression cassettes If inserted into the form of a stable plasmid away from the plasmid, its expression plasmid Can contain DNA segments from 2 micron DNA containing the source of replication, or If a yeast strain without long DNA is used, it can contain the entire 2 micron DNA . In this case, the latter type of plasmid is preferred. For example, according to the present invention, Sumids contain complete 2 micron DNA in continuous form. That is, 2 micron DNA Once cut with a restriction endonuclease, the linear DNA is Ligation to other components of the vector. The restriction sites are REP1, REP2 and FLP genes, And the normal function of the ORI, STB, IR1 and IR2 sites of 2 micron DNA and its FLP A small site near the center of each inversion repeat (IR) where the recombinase acts A good "FLP recognition target" (FRT) site is selected to be maintained. Optionally, a restriction Positions are also selected so that the D gene of the 2 micron DNA maintains its integrity. Appropriate Restriction sites include, for example, a unique Pst1 site located in the D gene, And unique Hpa1 and SnaB1 sites located all outside of the site. But Et al. Mentioned earlier in different (such as two) restriction sites, especially 2 micron DNA Insertion of an expression cassette and further components (e.g. Both are possible as well.   Such plasmid derivatives may contain only two inverted repeat FRT sites, or It may include a third FRT site. The former type of plasmid is hereinafter referred to as "symmetric 2 micron The latter type of plasmid is actually called a “hybrid vector”. Not only symmetric plasmids, but also symmetric 2 cells in yeast cells transformed with them. Despite generating a Klon-like hybrid vector, Ron-like non-integrated vector "(EP-A-501914).   The symmetric 2 micron-like hybrid vector of the present invention is preferably , The bacterial or viral DNA sequence, ie, the bacterial genome, plasmid or Does not contain virus-derived DNA. However, the 2 micron-like non-integral of the present invention The vector is a symmetric 2 micron-like hybrid vector generated from a non-integrated vector. Direct repeat FRs excised from a vector in transformed yeast cells It may include the prokaryotic DNA sequence between the T sites. These DNA sequences are described below. Sequence of the bacterium, such as the essential structural or functional characteristics of the vector. Or a symmetric 2 micron-like hybrid vector or a symmetric non-integral vector. In order to create a vector, an asymmetric 2 micron-like plasmid derivative or Fills two regions between the two inverted repeat FRT sites of a symmetric "non-integrated vector It may only have functions.   In a preferred embodiment, the inverted repeat FRT site in a circular form of 2 micron DNA The two regions between have suitably the same length.   Preferably, the expression plasmid according to the invention has one or more or in particular one or two tests. Selective genetic markers for the host used in, and such markers and Bacterial hosts (except symmetric 2 micron-like hybrid vectors), especially E. coli Includes replication sources for   Transformation for expression of marker gene phenotype as a selective genetic marker Any marker gene that facilitates selection for the body can be used. Appropriate Markers include, for example, those that express antibiotic resistance or auxotrophic host mutants. In the case of, it is a gene that complements the host disorder. The corresponding gene is, for example, the antibiotic G418 Resistant to nutrients, hygromycin or bleomycin, or nutrients Prototropy in auxotrophic yeast mutants, such asURA3,LEU2,LYS2,HIS3 OrTRP1Provide the gene.   Amplification of expression plasmids is convenient in prokaryotes such as E. coli As performed, for example, genetic markers for prokaryotes, such as E. coli genetic markers and prokaryotes, A source of replication for E. coli is advantageously included. These are replications of prokaryotes, such as E. coli. Includes genetic markers with resistance to sources and antibiotics such as ampicillin The corresponding prokaryotic plasmid, e.g. pBR322 or pUC plasmid, e.g. pUC18 Alternatively, it can be obtained from an E. coli plasmid such as pUC19.   According to the present invention, apart from the polypeptide expression cassette, the origin of replication and the genetic markers The expression plasmid contains additional expression cassettes such as 1-3 additional polypeptides. Arbitrarily.   Expression plasmids according to the present invention can be prepared by methods well known in the art. For example, a polypeptide expression cassette, for the host used for testing, and optionally A DNA fragment containing a selective genetic marker for a bacterial host; Source of replication for testing, and optionally for a bacterial host, and optionally Different functional fragments or expression cassettes by conventional chemical or biological assays. Can be prepared by coupling in a predetermined sequence using in vitro synthesis procedures . Preferably, the plasmid is made and prepared using recombinant DNA technology. set For preparation by recombinant DNA technology, the appropriate fragment is prepared in a test tube in a conventional manner. Connected within. The ligation mixture is then used to determine the appropriate nature of the modulator. Transformants that are transformed into prokaryotic or eukaryotic organisms and that contain the required vectors are commonly used. It is selected according to the general procedure. Plasmids are prepared by means of the transformed host. It can be handled and isolated in a conventional manner. The choice of host depends on the location of the vector. Depends on the knot array. The expression vector of the present invention is preferably a prokaryote, such as E. coli. And contain functional regulatory sequences, so that prokaryotic hosts, such as E. coli, Preferred for fabrication and handling.   Further embodiments of the invention include a signal sequence, a β-amyloid peptide or DNA encoding the functional derivative and a promoter operably linked to the terminator. Expression cassette containing a promoter and a hybrid plasmid containing the expression cassette Related. In a preferred form, the plasmid is a 2 micron S. cerevisiae. Based on plasmid.   Methods for making these expression cassettes from different functional fragments Are known in the art, for example, Sambrook et al., Molecular Cloning. : A laboratory manual, 2^nd Edn. Reported in 1989.   The transformed strain is cultured using methods well known in the art.   Corresponding complex culture media that can be used to culture yeast are known in the art. And are well known. For example, such culture media are tryptone, peptone, extract , Malt extract, yeast extract, casamino acid, corn steep solution, soy flour, whey , Whey hydrolysates and the like, especially mixtures thereof, and optionally further sugars (eg dex. Trose, glucose, sucrose, galactose, etc.), vitamins (eg, bio Tin), individual amino acids, inorganic salts (eg, sulfate, chloride, phosphate) Salts and carbonates of sodium, potassium, magnesium and calcium, furthermore And the corresponding salts of trace elements such as iron, zinc and manganese).   Take into account that all the essential elements shown above are present in the medium. Can be Preferred culture media are commercially available, optionally supplemented with inorganic salts and vitamins. Medium YPD (yeast extract, peptone, dextrose; for example, Methods Enzymol . 194, 13).   How to test   Another embodiment of the present invention is directed to the effects of compounds based on the appearance of abnormally folded proteins. A method for determining the transformed cells, as defined above, under appropriate conditions Cultivate the host, apply the compound to be tested, and reporter gene activity Measuring the amount of   This effect can be caused, for example, by abnormal folding of the protein or by incorrect folding of the protein. It can be based on the inhibition of conditions that promote tanning or prevent aggregation of the protein.   The test can be performed, for example, in the following form: Growing the transformed host with the two expression cassettes under appropriate conditions Step; • Optionally, a switch whose abnormal folding induces expression of the protein to be monitored. Tep, this induction occurs, for example, under the control of an inducible promoter that allows expression of this protein. Is necessary if Optionally, adding a compound that promotes abnormal folding or aggregation, which comprises Necessary if abnormal folding of the protein is due to additional compounds; Adding a test compound; and -Transcription or translation of reporter factors, or transcription or translation products or Steps to monitor for more provoked effects.   In a preferred embodiment of the present invention, the method comprises coagulating a β-amyloid peptide. Used for the identification of compounds that inhibit collection. These compounds are, for example, By binding to or modifying the protein, thereby inhibiting aggregation ; .Binds to or modifies proteins, thereby initiating or promoting protein aggregation By inhibiting the binding of other compounds that advance; or .Binding to or modifying a compound that initiates or promotes protein aggregation Than Can work.   Compound   New compounds and treatments identified using the methods of the invention, particularly those defined above The use of these compounds in methods of inhibiting protein aggregation is also included. This These new compounds can be used, for example, in the treatment of cancer or Alzheimer's disease. Brief description of drawings   In the following experimental section, various embodiments of the present invention are described with reference to the accompanying drawings. Listed:   FIG. 1 is a schematic diagram of the plasmid pCS2-1.   FIG. 2 is a schematic diagram of the plasmid pFL38 / CPY. Example   The following examples describe the invention in detail and should not be construed as limiting. .   Cleavage with restriction enzymes, ligation, transformation, annealing and β-galactosidase All enzymatic reactions, such as assays, are standard methods in genetic engineering, Essentially Sambrook et at ,. Molecular Cloning: A laboratory manual, 2^nd Ed n. Performed as described in 1989.   The enzyme used for all PCR reactions is Vent polymerase (New England Bio-L abs). Primers are synthesized by a DNA synthesizer.   Example 1: Unfolded protein response linked to a core promoter PPFY for integration of the lacz expression cassette into the yeast chromosome under the control of factors 7 Construction of vector It is a tar. It is a yeast selection markerSaccharomyces cerevisia D (Saccharomyces cerevisiae )LEU2Encodes a gene. It is CYClp-lacz fusion Also includes joint genes. CYClp is yeast iso-1-cytochrome C (CYC1) Gene A promoterlaczEncodes the E. coli β-β-galactosidase enzyme You.   Plasmid pPFY7 is obtained as follows. After digestion, the 2832 bp fragment was analyzed by agarose / TBE gel electrophoresis. (CA, USA).   About 2190bp Saccharomyces cerevisiaeLEU2The gene was transferred to plasmid pRS425 (AC TT 77106) as an Ssp1-Tth1 fragment. Digest pR.S425 with Tth1 After that, a large fragment of Klenow DNA polymerase is applied to the Partly with Ssp1 Purify more.   2832 bp Ssp1-Nac1 fragment and about 2190 bp Ssp1 flushed end The fragments are ligated and transformed into E. coli HB101. Obtained from individual transformants The obtained DNA is analyzed by restriction enzyme analysis. One with exact restriction fragment The clone is named pLEU2.   The plasmid pLEU2 is completely digested with XhoI and PvuII. About 4716 bp fragment Is isolated as described above. The XhoI-PvuII approximately 3500 bp fragment was first Digest with I and then partially with PvuII Upon digestion, plasmid pLG669-Z (Guarente & Ptashne, Proc. Natl. Ac ad. Sci. USA (1981), 2199-2203). Connect the two fragments Ligation and transformation into HB101. One clone with correct restriction fragment Named PFY7.   Example 2: Under the control of an unfolded protein response element linked to a core promoter Of a plasmid containing a Lacz expression cassette in Escherichia coli   22 bp unfolded protein response (UPR) factor (Mori et al., EMBO J (1992), 11, 25 83-2593) are chemically synthesized and have a double-stranded deoxyoligoribonucleotide linker. -(SEQ ID NO: 1). One of the fragments is an XhoI digested vector. XhoI sticky powder maintained as an XhoI site after subcloning into pPFY7 Including edges. Ligation is performed after linker tailing. SEQ ID NO: 1   Linker tailing includes the following steps.   About 100 n double-stranded deoxyoligoribonucleotides (see SEQ ID NO: 1) g of T4 DNA ligase (1 μl; 1 U / μl; Gibco-BRL, Basel, Suitzerland) Ligation to 1 μg of XhoI digested pPFY7 at 4 ° C. for 15 hours in the presence. The linked mixture Fractionation is performed by electrophoresis on a 1% agarose-Tris-acetate gel. Connected resources Including SA). Incubate the DNA at 95 ° C, then slowly to room temperature Cool and then reanneal by transforming into E. coli strain HB101. The DNA obtained from the transformant was added to 2% agaro. Analyze on gel. Clos showing a clear increase after digestion with XhoI and BamHI. Are those that have a UPR element in the correct orientation upstream of the CYC1 core promoter. You. One such plasmid is designated pCS2-1 (FIG. 1). The insertion, Ap Confirm by DNA sequencing using the plied Biosystems DNA Sequencer 370A. Admit.   A 22 bp unfolded protein response element (see SEQ ID NO: 1) (described above) L) Relink to pCS2-1 by linker tailing. This is two of the UPR factors Produces copy-encoding plasmid pCS2-2 and confirms it by DNA sequencing (See above).   The plasmids pCS2-3, pCS2-4 and pCS2-5 are obtained similarly. These are 3, 4 and Encodes 5 UPR factors. Confirm all insertions by DNA sequencing (see checking).   Example 3: Chromosome integration of UPR-CYClp-lacz fusion gene in yeast strain   Electroporation (Becker & Guarente, Methods-Enzymal, (1991), 194 182-187) for the transformation of yeast cells. Yeast strain W3116 (for all integration of p-lacz gene fusionMetaleu2-3  Leu2-1 12   his3  ura3-52  pcrl- Δ :: HIS3  pep4- Δ1137) (J. Winther, Carlsberg L aboratory, Denmark). Using the plasmid pJW1137, gene replacement (wins ton et al., Methods Enzymol. (1983) 1101, 211-228), van den Haze l et al., Eur. J. Biochem. (1992), 207, 277-283, strain W309. Prepare W3116 from 4. This plasmid contains the promoter and open-ready With the deletion of EcoRI to the ClaI restriction site, which obtained the first 75% of the ToPEP4Gene (Ammerer et al. , Mol. Cell. Biol. (1986), 6, 2490-2499).   The UPR-CYC1p-lacz construct (pCS2-1-5) was used to linearize the plasmid with BstEII followed by B stEII truncated strain W3116LEU2Integrate into the seat. Precise gene integration and gene replacement Is confirmed by PCR. Next, the amplified fragment is subjected to agarose gel electrophoresis. Analyze by   Example 4 Small Induced by Glycosylated Inhibitor Tunicamycin Abnormal protein folding in the endoplasmic reticulum induces the UPR-CYClp-lacz gene.   All yeast strains in Table 1 were grown at 30 ° C. for 24 hours in SD medium (0. Grow as pre-culture in 67% yeast nitrogen base, 2% glucose). Step An aliquot of the culture (1%) is transferred to YPD medium (1% Bacto yeast extract, 2% Bacto peptone, 2% glucose) Seed and grow cells at 30 ° C. for 16 hours (control). Induction of abnormal protein folding For guidance, aliquots of metaphase log phase cultures were incubated at 30 ° C. for 6 hours with tunicamycin (1 0 μg / ml; Sigma). The cells are collected and washed with 0.9% NaCl. 420m at m as o-nitrophenyl-β-D-galactopyranomide hydrolysis The measured β-galactosidase activity is normalized to the cell culture density, and To express. With plasmid pPFY7 (not treated with tunicamycin) The activity of the control strain is set to a value of 1. Three individual components (each assay is duplicated) Table 2 shows the results which are the average of the results of the experiments.   Example 5: 2 micron plasmid encoding expression cassette for wild-type CPY Production   Plasmid pLV9 is described in Vals ee al., Cell (1987), 48, 887-897. Complete encoding yeast carboxypeptidase Y enzyme (CPY) which is interpreted as WhatPRC1Including genes. Code for promoter (prepro-CPY from pLV9 ClaI-HindIII PRCI fragment The two micron-based vectors pDP34 (DSM 4473) and centromere SalI-SacI flag for convenient cloning into vector pFL38 (ATCC 77203) Transfer (5 'to 3').PRC1The operation of the restriction sites at 5 'and 3' Performed using SalI and SacI linkers available from Boehringer.   Plasmid pDP34 contains the complete S. cerevisiae 2-micron plasmid, As yeast selectable markerS. Cerevisiae URA3as well asdLEU2Encodes a gene E. coli-S. Cerevisiaes shuttle vector (EP-A-340170).   Plasmid pFL38 is also a centromereCEN6Autonomous copy distribution from S. cerevisiae Column (ARSS. cerevisiae as yeast selection markerURA3Code E. coli-S. Cerevisiae shuttle vector.   SalI-SacI in pDP34 (completely digested with SalI and SacI)PRC1Genetic After fragment subcloning, one correct plasmid is designated pDC13. You.   SalI-SacIPRC2The fragment was also digested with SalI and SacI to obtain pFL38 / CPY. Subclones into fully digested pFL38.   Example 6: Preparation of mutant CPY precursor gene   The mutations incorporated into the C-terminus of the CPY polypeptide sequence are shown in Table 3. Mature CPY The precursor probe CPY is used to form Sites treated with Nase B are indicated by the single letter notation N and K in Table 3. ) Prepro-CPY between residues Asn111 and Lys112. Wild type (wt) sequence Substituted residues are underlined.   Included in pFL38 / CPY (Valls et al., Cell (1987), 48, 887-897)PRC1157bp  Performing PCR-mediated site-directed mutagenesis based on the MunI-BamHI fragment Mutation is performed by   Sal-MunI fragment from pFL38 / CPY (see FIG. 2 and Example 5) and And the mutated MunI-BamHI fragment (see above) are completely eliminated with SalI and BamHI. Resulting plasmid, first subcloned into cloned pUC19 IsPRCMutations containing a 1159 bp SalI-BamHI fragment belonging to the 5 'end of 1 Is performed by DNA sequencing (see Example 2).   Example 7: 2 encoding expression cassette for mutant CPY precursor gene Preparation of micron plasmid   Complete encoding promoter, coding sequence and transcription terminator Using the mutant gene as a SalI-SacI fragment Bull.   (As subcloned in pUC19; including mutant prosequences of CPY (see See Example 7)) SalI-BamHI fragment, BamHI-NcoI (931 bp) and NcoI-Sac I (546 bp) (the latter two fragments are obtained from pFL38 / CPY; FIG. 1 and Example : 5) subcloned into pDP34 completely digested with SalI and BamHI. To synchronize. The resulting plasmid was designated pDC8 (encoding Mut1 PRC1), Named pDC9 (encoding ut2 PRC1) and pDC10 (encoding Mut3 PRC1). They are used for expression in yeast.   Example 8: Mutant PRC2 gene expresses abnormally folded protein.   Plasmid (MutlPRC1Code) pDC8, (Mut2PRCCode 1) pDC9 , (Mut3PRC1PDC10 and (wtPRC1Code) pDC13, element Stock W3116-3 × by kutroporationlaczTransformation intoPRC1gene Β-galactosidase expression induced by wild-type gene (wt-gene) Compare with the strain you have. Three individual transformations from each of the previous four transformants The body is grown as in Example 4. Each assay is performed in duplicate. Table 4 shows the results Shown in wtPRC1Β-galactosidase activity in strain YDC13 expressing Cut.   Similar results were obtained with strain W3116-4xlaczHas been obtained.   Example 9: Mutant pro-CPY protein has a lacz reporter with a complete KAR2 promoter It can also be monitored as an abnormal fold when under control of a monitor.   To make a KAR2p-lacz construct,CYC1XhoI-BamHI flag containing promoter Was removed from plasmid pCS2-1 and the 630 bp SalI-BamHIKAR2promoter Replacement with a fragment (Rose et al., Cell (1989), 57, 1223-1236).K AR2 The promoter was the two primers of SEQ ID NOs: 6 and 7. By PCR Isolate. The template for PCR is yeast strain S288C (ATCC26108). The resulting plasmid is named pCSFOL4. This is described in Example 3. And integrated into W3116. Confirm correct integration (see Example 3) thing). One of these strains is W3116KAR2p-laczIs referred to as   Plasmid (Mut1PRC1Code) pDC8, (Mut2PRC1Code) pDC9 , (Mut3PRC1PDC10 and (wtPRC1Elect pDC13 Stock W3116 by ROPKAR2p-lacz(See above).   MutationPRC1Β-galactosidase expression induced by the gene Compare with the strain carrying the gene. Three individual from each of the previous four transformants Transformants are grown as in Example 4. Each assay is performed multiple times. That Table 5 shows the results. wtPRC1Β- in a strain transformed with pDC13 encoding Set galactosidase activity to a value of 1.   Example 10: Human β-amyloid peptide without any signal peptide (β Preparation of expression cassette for A4)   (Encoding only βA4 without signal sequence (expression isGAPCLPromoter control The plasmid pJC21 is made as follows.   (About 275 bp SalI-BamHI pBR322 fragment and yeastGAPDHFrom the gene SalI-BgIII fragment of GAPCL) Is isolated by PCR using the primers of SEQ ID NOs: 8 and 9. Chaudhuri e t al. Eur. J. Biochem. (1992), 206, interpreted as described in 793-800 Plasmid pBC2 is used as a template. v. Biochem. (1989), 58, 287-307) was prepared as SEQ ID NO: 10/1. Chemical synthesis using one oligomer and two Amplify by PCR using primers (SEQ ID NOs: 12 and 13). BgIII-Ec of βA4 The oRI fragment was subcloned into pUC19BgI (the BamHI site of pUC19 was The III site was modified to form pUC19Bgl; the BamHI site was digested with Klenow polymerase. BgIII linker / Boehringer is ligated, then digested with BgIII and ligated again The sequence is confirmed in Example 2. SEQ ID NO: 10/11   EastSUC2Genes (Taussig & Carlson, Nucleic Acids. Res. (1983), 11 EcoRI-SacI terminator fragment from SEQ ID NOs: 14 and 1954). And PCR with 15 primers. Ys (from wild-type strain S288C) Genomic DNA is used as a template.   The three fragments (SalI-BgIII, BgIII-EcoRI and EcoRI-SacI) were: pJC21 (see Table 6) was subcloned with pDP34 completely digested with alI and SacI. ) Is generated.   Example 11: of human β-amyloid peptide (βA4) having a signal peptide Of expression cassette for   (Bound to βA4SUC2Encodes a signal sequence; expression isGAPCLpromoter PJC22 (under the control of) bound to βA4PRC1Encodes a prepro sequence; Expression isPRC1Under the control of a promoter) pJC26 and (nuclear localization sequence from SV40 large T antigen sequence, NLS, and βA4 Do: expression isGAPCLPDP34-NLS-βA4 under the control of pJC21 (Example 10; see Table 6).   SVC2Bound to signal sequenceGAPCLSalI-BgIII fragment containing promoter PCR (ie, pBR322-GAPCLp-lnvss) using the primers of SEQ ID NOs: 8 and 16 Amplify by Using plasmid pBC1 (see Example 10) as template You.   Bound to the prepro sequence of CPYPRC1SalI-BgIII flag including promoter PCR using the primers of SEQ ID NOs: 17 and 18 Amplify by The template is pLV9 (see Examples 5 and 6) ).   SalI- containing the GAPCL promoter linked to the nuclear localization sequence from the SV40T antigen The BgIII fragment (ie, pBR322-GAPCLp-NLS) was replaced with the primers of SEQ ID NOs: 8 and 19. The template for PCR is approximately 275b from pBR322. p SalI-BamHI fragment, about 400 bp BamHI-EcoRI GAPCLp fragment Encoding the EcoRI-SpeI fragment of the nuclear localization sequence from the SV40T and SV40T antigens Rasmid pRH3. This nuclear localization sequence (Nelson & Silver, Mol. Cell. Biol. (1989) 9,384-389) uses the deoxyoligoribonucleotide of SEQ ID NO: 30. And is chemically synthesized. SEQ ID NO: 30   One of the previous SalI-BgIII fragments, (encoding βA4 (see Example 10)) Bg III-EcoRI fragment and (SUC2Code the terminator (see Example 10) The EcoRI-SacI fragment was digested with pD (completely digested with SalI and SacI). Subcloning into P34 to generate pJC22, pJC26 and pDP34-NLS-βA4, respectively .   Example 12: Transformants pJC21, pJC22, pJC26 and pDP34-NLS-βA4 and β-gal Yeast Transformation of Cuctosidase Assay   Transform the plasmid into strain W3116-3xlacz(See Example 3). β-Ga Lactosidase activity is measured (as in Example 4). See Table 7.   Example 13: Preparation of C-terminal truncation of βA4; yeast transformant; β- Galactosidase assay   Two plasmids (SUC2Truncated form of signal sequence and βA4, 1-39 PJC24 and (code)SUC2Signal sequence and βA4 truncated form, 1-3 PJC25 (encoding 6) is made as described below. Both expression cassettesGA PCL Under the control of a promoter. Peptide βA4,1-39And βA4,1-36Is a trial It has a slower aggregation rate in vitro (Burdick et al., J. Biol. Chem. (1992), 267, 546-554; Barrow et al., Mol. Biol. (l992), 225, 1075- 1093; Pike et al. Neuroscience (1993), 13, 1676-1687).   Two SalI-XbaI fragments, pBR322-GAPCLp-Invss-βA41-39And pBR32 2-GAPCLp-Invss-βA4, 1-36 was converted to two primers (SEQ ID NO: 8 and 20 and, for the latter construct, SEQ ID NOs: 8 and 21) by PCR. Isolated. The template is pJC22.   EastSUC2The XbaI-SacI terminator fragment from the gene was By PCR using yeast genomic DNA (from wild-type strain S288C) as a plate Amplify. The primer has SEQ ID NO: 22 and 15.     SalI-XbaI fragment and XbaI-SacI fragment were digested with SalI and SacI. Subclones into fully digested pDP34 (see Table 8).   Transform the plasmid into strain W3116-3xlacz(See Example 3). β-gala Cactosidase activity was measured (as in Example 4) and the strain YJC2 described in Example 10 was tested. Compare with 1 and YJC22.   Example 14: Preparation of rat mutant βA4 (mrβA4) and inverted human βA4 (iβA4)   Plasmids pJC21 and pJC28 are interpreted as shown in Table 10. Both expression cassettes IsPRC1Contains the prepro sequence of the gene and is driven by the PRC1 promoter. pJ C27 is a rat mutant βA4 (mrβA4) (Dyrks et al., FEBS. Lett. (1993), 324, 231). -236; Hilbich et al., Mol. Biol: (1991), 218, 149-163), and pJC2 8 is inverted human βA4 (iβA4) (Fraser et al., Biochemistry (1992), 31, 10716-107). Code 23).   As described in Table 10, the plasmid contains the CPY promoter and the pre- SalI-BgIII fragment (including either mrβA4 or iβA4) B gII-EcoRI and EcoRI-SacI fragment of SUC2 terminator You.   The BgIII-EcoRI fragment (containing either mrβA4 or iβA4) has two Overlapping oligodeoxyribonucleotides (SEQ ID NOS: 25/26 and 27/28 And fragment 5 Two primers that hybridize to the 3 ′ and 3 ′ ends (see SEQ ID NOs: 12 and 13) ).   SalI-BgIII, BgIII-coRI and EcoRI-SacI fragments were ligated with SalI and Sac Subclones into pDP34 completely digested with I.   Table 11 shows the amino acid sequences of βA4, mrβA4, and iβA4. wt human βA4 sequence Differences are underlined. SEQ ID NO: 23/24 SEQ ID NO: 25/26   Plasmids in Table 10 were transformed into strain W3116-3xlaczTransformation (see Example 3) Replace. β-galactosidase activity was measured as in Example 4 and reported in Example 10. Compare with listed strains YJC26, YJC21 and YJC22 (see Table 12).   Example 15: Trivalent aluminum and divalent zinc fold βA4 abnormally in yeast Increase folding.   Cation Al³⁺And Zn²⁺Is known to induce βA4 aggregation in vitro (Mantyh et al., J. Neurochemistry (1993), 61, 1171-1174). β-galacto The activity of AIs_Three(About 3 mM; Merck) and ZnCl_Two(About 300 μM; Merck) The cells are grown in YPD as described above and then measured as in Example 4. The results are shown in Table 13 . β-GA represents β-galactosidase activity. The symbol "-ive control" indicates that the cells It means that it proliferated in the absence of either of the two transition metal cations.   Example 16: Desferal and ascorbic acid reduce abnormal folding of βA4 You. Minimal medium SD in the absence or presence of IBA-GEIGY) and ascorbic acid (Merek) (Example 4). β-galactosidase activity was determined as in Example 4. To be measured. The results are shown in Tables 14 and 15. β-GA shows β-galactosidase activity You. The symbol "-ive control" indicates that the cells are desferrioxamine or ascorbic acid. Means that they grew in the absence of any of the above.   DFO and ascorbic acid both cause abnormal βA4 folding in yeast cells / It appears to reduce agglomeration. Deposit   The following microbial strains were used by Deutsche Sammlung von Mikroorganismen (DSM), Maschero der Weg 1b, D-38124 Braunschweig (Accession number and date of deposit are as follows)   E. coli JM109 / pDP34 DSM4473 March 14, 1988

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Claims (1)

[Claims] 1. A host transformed with at least a first and a second expression cassette, wherein the first expression cassette comprises one or more unfolded protein responsive elements (UPRs) operatively linked to a reporter element; A second expression cassette comprising a signal sequence, a DNA encoding the protein whose abnormal folding is to be monitored, and a promoter operably linked to a terminator, wherein the first and second expression cassettes are in a host. Wherein the protein to be monitored in said second expression cassette is selected from the group consisting of prions, p53, β-amyloid peptide and functional derivatives thereof. . 2. The host according to claim 1, which is capable of secreting a protein. 3. The host according to claim 1, which is a plant, an insect, a mammal, a fungus, or an animal cell. 4. 2. The host according to claim 1, wherein the host is a fungal cell. 5. The host according to claim 1, which is a yeast cell. 6. 2. The host according to claim 1, wherein the host is Saccharomyces cerevisiae. 7. The host according to claim 1, wherein the UPR of the first expression cassette is derived from BiP. 8. 2. The host of claim 1, wherein the UPR of the first expression cassette comprises the DNA sequence set forth in SEQ ID NO: 1 or a functional equivalent thereof. 9. The host of claim 1, wherein said UPR is present in one or more replicates in said first expression cassette. Ten. The host of claim 1, wherein the UPR is present in the first expression cassette in 2 to 5 replications. 11． The host of claim 1, wherein said reporter element comprises said promoter operably linked to DNA transcribed under control of said promoter and to a terminator. 12． 12. The host of claim 11, wherein said promoter in said first expression cassette is tightly controlled. 13. 13. The host according to claim 12, wherein said promoter is selected from the group consisting of CYC1 and KAR2 promoters. 14. 2. The host of claim 1, wherein transcription of the reporter factor causes an effect that can be easily measured during or after transcription or translation. 15． The host according to claim 1, wherein the reporter element encodes a protein whose amount can be easily determined. 16． 16. The host according to claim 15, wherein said reporter protein is selected from the group consisting of β-galactosidase and luciferase. 17． 12. The host according to claim 11, wherein the terminator is selected from the group consisting of a terminator naturally binding to the transcribed DNA, PHO5 , α-factor, and a SUC2 gene terminator. 18． The host according to claim 1, wherein the promoter of the second expression cassette is an inducible promoter. 19. The promoter of the second expression cassette, wherein the promoter originally linked to the DNA encoding the protein whose abnormal folding is to be monitored, CUP1, GAPDH, GAPFL, GAL (10), PYK, TPI, ADH, PRC1, and PG The host according to claim 1, wherein the host is selected from the group consisting of a K promoter. 20. The host according to claim 1, wherein the promoter of the second expression cassette is a GAPFL promoter or a functional derivative thereof. twenty one. The host according to claim 1, wherein the signal sequence of the second expression cassette is selected from the group consisting of SUC2, α-factor, KEX1, PHO5, and glucoamylase signal sequence. twenty two. The host according to claim 1, wherein the signal sequence of the second expression cassette is SUC2 or α-factor signal sequence. twenty three. 2. The host of claim 1, wherein the protein to be monitored in said second expression cassette has an abnormal amount of hydrophobic groups on its surface. twenty four. The host of claim 1, wherein the protein to be monitored in the second expression cassette is or comprises a β-amyloid peptide or a functional derivative thereof. twenty five. The host of claim 1, wherein the terminator of the second expression cassette is selected from the group consisting of a terminator naturally adjacent to DNA encoding the protein, an alpha factor terminator, and a SUC2 terminator. 26. The expression cassette according to claim 1, comprising a signal sequence, a DNA encoding a β-amyloid peptide or a functional derivative thereof, and a promoter operatively linked to a terminator. 27. A hybrid plasmid comprising the expression cassette according to claim 26. 28. 28. The hybrid plasmid according to claim 27, which is based on a 2 micron plasmid of S. cerevisiae. 29. A method for determining the effect of a compound on the appearance of an abnormally folded protein, comprising culturing the transformed host of claim 1 under appropriate conditions, and applying the compound to be tested. And measuring the amount of reporter gene activation. 30. Growing the transformed host of claim 1 under appropriate conditions; optionally, inducing expression of the protein whose abnormal folding is to be monitored; and optionally, causing abnormal folding or aggregation. Adding a promoting compound; adding the test compound; and monitoring the transcription or translation of the reporter factor, or a transcription or translation product or an effect caused thereby. Item 30. The method according to Item 29. 31. 30. The method according to claim 29 for the identification of a compound that inhibits aggregation of β-amyloid peptide. 32. A compound identified using the method of claim 29. 33. Use of a compound according to claim 32 in a method of treatment. 34. Use of a compound according to claim 32 for the inhibition of protein aggregation. 35. Use of a compound according to claim 32 for the treatment of Alzheimer's disease.