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WO1996008561A1 - Procede de detection de proteines mal repliees - Google Patents

Procede de detection de proteines mal repliees Download PDF

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Publication number
WO1996008561A1
WO1996008561A1 PCT/EP1995/003475 EP9503475W WO9608561A1 WO 1996008561 A1 WO1996008561 A1 WO 1996008561A1 EP 9503475 W EP9503475 W EP 9503475W WO 9608561 A1 WO9608561 A1 WO 9608561A1
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WIPO (PCT)
Prior art keywords
promoter
expression cassette
host according
protein
feature
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PCT/EP1995/003475
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English (en)
Inventor
Bhabatosh Chaudhuri
Christine Stephan
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Novartis Ag
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Application filed by Novartis Ag filed Critical Novartis Ag
Priority to AU35215/95A priority Critical patent/AU3521595A/en
Priority to EP95931986A priority patent/EP0783574A1/fr
Priority to JP8509869A priority patent/JPH10506271A/ja
Priority to FI971034A priority patent/FI971034L/fi
Publication of WO1996008561A1 publication Critical patent/WO1996008561A1/fr
Priority to MXPA/A/1997/001881A priority patent/MXPA97001881A/xx

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the current invention concerns a new method for the detection of malfolded proteins; a method for the measurement of the amount of malfolded protein produced; and a method for the detection of compounds that influence the malfolding.
  • the biological function of a protein is governed by specifically adopted three-dimensional structures. Malfolded or aggregated proteins fail to acquire the necessary conformation required for the activity of a protein. Usually, the accumulation of malfolded proteins in the cell is connected directly or indirectly to several diseases like Alzheimer's disease (AD) and human prion associated diseases like Creutzfeldt-Jacob disease and Gertsmann- Straussler-Scheinker disease.
  • AD Alzheimer's disease
  • human prion associated diseases like Creutzfeldt-Jacob disease and Gertsmann- Straussler-Scheinker disease.
  • Another example for the effect of a malfolded protein is the tumor supressor protein p53 that is a negative regulator of cell growth (Donehower & Bradley, Biochim. Biophys. Acta (1993), 1155, 81-205).
  • the majority of human tumors express mutant p53 proteins. Hence, it is believed, that mutant p53 proteins are malfolded, or at least partially denatured.
  • Alzheimer's Diseases is pathologically connected to the appearance of cerebrovasular amyloid deposits.
  • the major protein found in these deposits is a 39-42 amino acid peptide named ⁇ -amyloid peptide mainly in the form of cross ⁇ -conformation.
  • APP amyloid precursor protein
  • APP amyloid precursor protein
  • the influence of compounds on the malfolding of proteins can be monitored via an easily measurable reporter gene product if the DNA coding for the malfolded protein is connected operable to a signal sequence that induces the transport of this protein through the endoplasmic reticulum (ER) and if one or more UPR elements are connected operable to the reporter element.
  • the inventive method also provides an easy method for determining whether unusual aggregation of a protein is related to malfolding or not. For example in the case of the ⁇ -amyloid peptide it has been surprisingly found, that aggregation that occurs in vivo and in vitro is associated to malfolding and can be monitored in vivo using the inventive system.
  • the rate of aggregation of different ⁇ -amyloid peptides (shortened, inverted or from different organisms) as estimated in vitro is closely correlated to the rate of malfolding measured with the inventive system. Therefore, it is also possible to monitor the influence of mutations on the rate of malfolding and the effect associated therewith.
  • the present invention concerns a host transformed with at least a first and a second expression cassette, wherein
  • the first expression cassette comprises one or more unfolded-protein-response elements (UPR) operably linked to a reporter element
  • the second expression cassette comprises a promoter operably linked to a signal sequence, to a DNA encoding a protein whose malfolding is to be monitored and to a terminator; and wherein these first and second expression cassettes are not naturally occurring in the host and wherein the protein to be monitored in the second expression cassette is selected from the group consisting of a prion, p53, ⁇ -amyloid peptide and functional derivatives thereof .
  • a suitable host may be any host which is capable of an 'unfolded-protein response', like hosts that secrete proteins via the ER.
  • suitable hosts are plant, insect, mammalian, fungal, or animal cells. Preferred are fungal cells, more preferred is a yeast cell and most preferred is Saccharomyces cerevisiae.
  • Suitable yeast strains according to the invention include strains of S. cerevisiae containing the endogenous two-micron plasmid or such strains which have been cured of said endogenous two-micron plasmid (see EP-A-340170).
  • the host can be transformed with the required first and second expression cassette by means commonly used in genetic engineering and as described, for example, in Sambrook etal., Molecular Cloning: A laboratory manual, 2 nd Edn. 1989.
  • An unfolded-protein-response elements is a fragment of DNA that is involved in the transcription initiation in case malfolded protein accumulates.
  • UPR elements can be isolated, for example, from the initiation region of a protein that is involved originally in the response to the accumulation of malfolded protein as for example from BiP, FKB2, BiP/GRP78, GRP94, PDI/ERp59 and ERp72 (Shamu et al., Trends in Cell Biol. (1994), 4, 56-60).
  • a preferred UPR is isolated from BiP and more preferably comprises a DNA sequence as depicted in SEQ ID NO 1 or is a functional equivalent thereof.
  • Functional equivalent has the meaning of a DNA sequence that is derived from SEQ ID NO 1 by replacement of some of the restriction sites at one or both ends or by replacement of nucleotides, e.g. 1 to 5, that do not interfere with the activity to respond to the accumulation of unfolded protein.
  • the UPR may be present in more than one copy, for example in 2 to 5 copies.
  • the reporter element comprises a promoter operably linked to a DNA that is transcribed under the control of this promoter, and to a terminator.
  • the promoter in this reporter element can be of almost any origin. It is for example possible to use a tightly regulated promoter or the promoter that is naturally adjacent to said DNA, like the ⁇ -galactosidase or luciferase promoter, or the CYC1, the Gal1/GAL19, PH05 or the KAR2 promoter. In a preferred embodiment of the invention the promoter is selected from the group consisting of the CYC1 and the KAR2 promoter.
  • a suitable DNA that is transcribed under the control of this promoter usually causes an effect that can be measured easily during or after transcription or translation.
  • transcription or translation products that can be measured easily e.g. via the measurement of the amount of protein, mRNA or DNA produced; or that cause an effect that can be measured easily, e.g. cell growth, an enzymatic reaction or color.
  • the transcribed DNA codes, for example, for a protein that is produced in an amount that is related to the amount of activation of said promoter and that is, e.g., not produced elsewhere in the chosen host under the applied conditions.
  • suitable proteins are the metailothionein that is encoded by the yeast CUP1 gene, ⁇ -galactosidase or luciferase. Preferred is luciferase and ⁇ -galactosidase.
  • a suitable terminator for this first expression cassette usually contains also the proper signals for transcription termination.
  • This terminator may be naturally linked to the transcribed DNA or may be introduced from a different origin like the PHQ5. the ⁇ -factor or the SUC2 terminator.
  • This first expression cassette may, for example, contain additionally a DNA sequence encoding a signal peptide as defined below.
  • the promoter for the second expression cassette according to the invention can be almost any promoter homologous or heterologous to the host. It is, for example, possible to use the promoter originally linked to the DNA encoding the protein whose malfolding is to be monitored; or it can be promoter commonly used in genetic manipulations of the host.
  • the promoter can be inducible or not inducible like the CUP1 , GAPDH, GAPFL, GAL(1/10), PYK, TPI, ADH, PRC1 and PGK promoter. Preferred is the GAPFL promoter or a functional derivative thereof.
  • the host can be grown under optimal growth conditions and the expression of the protein whose malfolding is to be monitored can be induced at a desired moment.
  • the signal sequence usually is chosen in accordance with the host that is used in the test.
  • a suitable signal sequence can be derived from any gene coding for a polypeptide that is ordinarily secreted. Examples are the SUC2, CPY, ⁇ -factor, KEX1 , PH05 and the glucoamylase signal sequence.
  • the protein whose malfolding is to be monitored are prions, p53, the ⁇ -amyloid peptide or functional derivatives thereof.
  • the protein to be monitored comprises the ⁇ -amyloid peptide or a functional derivative thereof.
  • the expression functional derivative denotes a peptide that is derived from the original malfolded peptide by minor changes, for example through up to 10 amino acid replacements, addition, or deletion, but wherein the properties in respect to malfolding are comparable or equal.
  • the terminator used in the second expression cassette can be the terminator naturally adjacent to the DNA coding for the protein whose malfolding is to be monitored, or another terminator as described above.
  • the promoter, the DNA sequence coding for the signal peptide, the DNA sequence coding for the polypeptide and the DNA sequence containing transcription termination signals are operably linked to each other, i.e. they are juxtaposed in such a manner that their normal functions are maintained.
  • the array is such that the promoter effects proper expression of the signal sequence-polypeptide gene complex, the transcription termination signals effect proper termination of transcription and polyadenylation and the signal sequence is linked in the proper reading frame to the polypeptide gene in such a manner that the last codon of the signal sequence is directly linked to the first codon of the gene for the polypeptide.
  • the promoter is preferably joined to the signal sequence between the major mRNA start and the ATG naturally linked to the promoter gene.
  • the signal sequence has its own ATG for translation initiation.
  • the junction of these sequences may, for example, be effected by means of synthetic oligodeoxynucleotide linkers carrying the recognition sequence of an endonuclease.
  • the expression cassettes according to the invention may be inserted into the genome of the host or in form of a stable plasmid like the two micron plasmid. If one or both of the expression cassettes are inserted in form of a stable plasmid, apart from the polypeptide expression cassettes the expression plasmids can comprise a DNA segment originating from two-micron DNA containing the origin of replication or, if a two-micron DNA free strain of yeast is used, total two-micron DNA. The latter type of plasmids is preferred in this case.
  • the plasmids according to the invention contain the complete two-micron DNA in an uninterrupted form, i.e.
  • restriction site is chosen such that normal function of the REP1 , REP2 and FLP genes and of the ORI, STB, IR1 and IR2 sites of two-micron DNA as well as small "FLP recognition target” (FRT) sites, located near the center of each inverted repeat (IR) at which the FLP recombinase acts, is maintained.
  • FRT FLP recognition target
  • the restriction site is chosen such that the D gene of two-micron DNA is kept intact too.
  • Suitable restriction sites are, for example, the unique Pstl site located within the D gene and the unique Hpal and SnaBI sites located outside of all of said genes and sites.
  • Such a plasmid derivative may comprise only two invertedly repeated FRT sites or an additional, third FRT site.
  • the former kind of plasmid is hereinafter called a "symmetric two micron-like hybrid vector”.
  • the latter kind of plasmid is hereinafter called “symmetric two micron-like disintegration vector” despite it is not a real symmetric plasmid but gives rise to a symmetric two micron-like hybrid vector in the yeast cell transformed therewith (EP-A- 501 914).
  • a symmetric two micron-like hybrid vector of the invention does preferentially not contain bacterial or viral DNA sequences, i.e. DNA derived from a bacterial genome, plasmid or virus.
  • a two micron-like disintegration vector of the invention may comprise DNA sequences of prokaryotic origin between the two directly repeated FRT sites which are excised from the vector in the transformed yeast cell in which the symmetric two micron-like hybrid vector is generated from the disintegration vector.
  • DNA sequences are bacterial sequences as described below and can provide to the vector essential structural or functional features or can also only have the function of filling up the two regions between the two invertedly repeated FRT sites of an unsymmetric two micron-like plasmid derivative or of an "unsymmetric" disintegration vector in order to construct a symmetric two micron ⁇ like hybrid vector or a symmetric disintegration vector.
  • the two regions between invertedly repeated FRT sites of the circular form of the two-micron DNA have approximately the same length.
  • the expression plasmids according to the invention include one or more, espe ⁇ cially one or two, selective genetic markers for the host used in the test and such a marker and (except for symmetric two-micron like hybrid vectors) an origin of replication for a bacterial host, especially Escherichia coli.
  • any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene.
  • Suitable markers are, for example, those expressing antibiotic resistance or, in the case of auxotrophic host mutants, genes which complement host lesions.
  • Corresponding genes confer, for example, resistance to the antibiotics G418, hygromycin or bleomycin or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3. LEU2. LYS2. HIS3 or TRP1 gene.
  • a prokaryote such as E. coli
  • a prokaryote e.g. E. coli
  • genetic marker and a prokaryote e.g. E. coli, replication origin
  • a prokaryote e.g. E. coli, replication origin
  • corresponding prokaryotic plasmids for example E. coli plasmids, such as pBR322 or a pUC plasmid, for example pUC18 or pUC19, which contain both prokaryotic, e.g. E. coli, replication origin and genetic marker conferring resistance to antibiotics, such as ampicillin.
  • the expression plasmids according to the invention contain optionally additional expression cassettes, such as 1 to 3 additional polypeptide expression
  • the expression plasmids according to the invention are prepared by methods known in the art, for example by linking the polypeptide expression cassette, the DNA fragments containing selective genetic markers for host used in the test and optionally for a bacterial host, the origin(s) of replication for yeast and optionally for a bacterial host, and optionally additional functional fragments or expression cassettes in the predetermined order using conventional chemical or biological in vitro synthesis procedures.
  • the plasmids are constructed and prepared using recombinant DNA techniques.
  • suitable DNA fragments are ligated in vitro in conventional manner.
  • the ligation mixture is then transformed into a suitable prokaryotic or eukaryotic host depending on the nature of the regulatory elements used, and a transformant containing the desired vector is selected according to conventional procedures.
  • the plasmids can be multiplicated by means of the transformed hosts and can be isolated in conventional manner. The choice of the host depends on the regulatory sequences located on the vector.
  • the expression vectors of the invention preferentially comprise regulatory sequences functional in prokaryotes, e.g. E. coli, a prokaryotic host, e.g. E. coli, is preferred for the construction and multiplication of the vector.
  • a further embodiment of the invention concerns an expression cassette comprising a promoter operably linked to a signal sequence, a DNA encoding the ⁇ -amyloid peptide or a functional derivative thereof, and to a terminator, a hybrid plasmid comprising said expression cassette.
  • this plasmid is based on the two-micron plasmid of S. cerevisiae .
  • the transformed strains are cultured using methods known in the art.
  • Corresponding complex culture media which can be used for culturing yeast are known in the art.
  • such culture media contain tryptone, peptone, meat extracts, malt extracts, yeast extracts, casamino acids, corn steep liquor, soy bean flour, whey, whey hydrolysate etc., and especially mixtures thereof and are optionally additionally supple ⁇ mented with sugars (e.g. dextrose, glucose, sucrose, galactose etc.), vitamins (e.g.
  • a preferred culture medium is the commercially available medium YPD (yeast extract, peptone, dextrose; cf. Methods Enzymol. 194, 13) optionally supplemented with inorganic salts and vitamins.
  • YPD yeast extract, peptone, dextrose; cf. Methods Enzymol. 194, 13
  • Another embodiment of the invention concerns a method for the determination of the influence of a compound on the appearance of malfolded protein, comprising culturing a transformed host as defined above under suitable conditions, applying the compound to be tested and measuring the amount of reporter gene activation.
  • This influence may be based, for example, on the inhibition of conditions that induce malfolding of said protein, promotion of the correct folding of said protein or prevention of the aggregation of said protein.
  • test can be carried out, for example, in the following form:
  • the method is used for the identification of compounds that inhibit the aggregation of ⁇ -amyloid peptide.
  • These compounds can act, for example, by
  • new compounds identified using the inventive method and the use of these compounds in a method of treatment and especially for the inhibition of protein aggregation as defined above. These new compounds may be used, for example, in the treatment of cancer or Alzheimer's disease.
  • Fig. 1 is a schematic illustration of plasmid pCS2-1.
  • Fig. 2 is a schematic illustration of plasmid pFL38/ CPY.
  • the enzyme employed for all PCR reactions is Vent polymerase (New England Bio-Labs).
  • the Primers are synthesized on a DNA synthesizer.
  • Example 1 Construction of pPFY7 vector for integration of the lacz expression cassettes. under the control of the unfolded-protein-response element(s) linked to a core promoter, into the veast chromosome
  • the plasmid pPFY7 is a pBluescript (Stratagene ® ) based vector. It encodes the Saccharomyces cerevisiae LEU2 gene as a yeast selection marker. It also contains the CYC1p-lacz fusion gene. CYC1p represents the core promoter of the yeast iso-1- cytochrome c (CYC1) gene and lacz encodes the E. coli ⁇ -galactosidase enzyme.
  • CYC1p represents the core promoter of the yeast iso-1- cytochrome c (CYC1) gene and lacz encodes the E. coli ⁇ -galactosidase enzyme.
  • the plasmid pPFY7 has been obtained as follows.
  • the plasmid pBluescript SK+ (Stratagene ® ) is digested with Sspl and Nael and a 2832 bp fragment is isolated by agarose/TBE gel electrophoresis. The fragment is isolated and purified by GeneClean ® (Bio 101, CA, USA).
  • the -2190 bp Saccharomyces cerevisiae LEU2 gene is isolated as a Sspl-TthI fragment from the plasmid pRS425 (ACTT 77106). After digesting pRS425 with Tthl, the sticky end is flushed with the large fragment of Klenow DNA polymerase which is followed by a partial digest with Sspl. The above fragment is isolated and purified by GeneClean ® as above.
  • the 2832 bp Sspl-Nael fragment and a -2190 bp Sspl-flushed end fragment are ligated and transformed in E. coli HB101.
  • DNA obtained from individual transformants are analyzed by restriction enzyme analysis.
  • One clone with the correct restriction fragments is named pLEU2.
  • the plasmid pLEU2 is completely digested with Xhol and Pvull.
  • a -4716 bp fragment is isolated as described above.
  • An Xhol-Pvull -3500 bp fragment is isolated from the plasmid pLG669-Z (Guarente & Ptashne, Proc. Natl. Acad. Sci. USA (1981), 2199-2203) by first digesting with Xhol and then performing partial digestion with Pvull. The two fragments are ligated and transformed in HB101.
  • One clone with the correct restriction fragments is named pPFY7.
  • Example 2 Construction of plasmids containing lacz expression cassettes, under the control of the unfolded-protein-response element(s) linked to a core promoter
  • the 22 bp unfolded-protein-response (UPR) element (Mori et al., EMBO J (1992), 11, 2583- 2593) is chemically synthesized and consists of double-stranded deoxyoiigoribonucleotide linkers (SEQ ID NO. 1).
  • the fragment contains Xhol sticky ends, one of which is maintained as a Xhol site after subcloning in the Xhol digested vector pPFY7. Ligation is performed after linker-tailing.
  • Linker-tailing involves the following steps. -100 ng of double-stranded deoxyoligoribonucleotides (see SEQ ID NO 1) is ligated to 1 ⁇ g of Xhol digested pPFY7 at 4°C for 15 h in the presence of T4 DNA ligase (1 ⁇ l; 1U/ ⁇ l; Gibco- BRL, Basel, Switzerland). The ligation mixture is fractionated by electrophoresis on a 1% agarose-Tris-acetate gel. The linear fragment, which contains the ligated linkers, is isolated and purified by GeneClean ® (Bio 101 , CA, USA).
  • the DNA is re-annealed by incubation at 95°C followed by slow cooling to room temperature and is then transformed in the E. coli strain HB101.
  • the DNA obtained from the transformants is analyzed on a 2% agarose gel.
  • the clones which show a distinct increase after digestion with Xhol and BamHI are the ones which have the UPR element in the correct orientation upstream of the CYC1 core promoter.
  • One such plasmid is named pCS2-1 ( Figure 1).
  • the insert is confirmed by DNA sequencing using the Applied Biosystems DNA sequencer 370A.
  • the 22 bp unfolded-protein-response element (see SEQ ID NO 1) is ligated to pCS2-1 , again via linker-tailing (as described above). This yields the plasmid pCS2-2 which encodes two copies of the UPR element and is confirmed by DNA sequencing (see above).
  • the plasmids pCS2-3, pCS2-4 and pCS2-5 are obtained similarly. These encode three, four and five UPR elements, respectively. All inserts are confirmed by DNA sequencing (see above).
  • the Bio-Rad gene pulser ® is employed for transformation of yeast cells by electroporation (Becker & Guarente, Methods-Enzymol, (1991 ), 194, 182-187).
  • the yeast strain W3116 ( ata leu2-3 Ieu2-112 his3 ura3-52 ⁇ cr1- ⁇ :: HI S3 Deo4- ⁇ 1137 ⁇ (J. Winther, Carisberg Laboratory, Denmark) is used for all integrations of UPR-CYC1p-lacz gene fusions into the yeast chromosome.
  • W3116 is constructed from the strain W3094 as described in van den Hazel et al., Eur. J. Biochem.
  • This plasmid carries a PEP4 gene (Ammerer et al., Mol. Cell. Biol. (1986), 6, 2490-2499) with a deletion from the EcoRI to the Clal restriction site, which takes out the promoter and first 75% of the open- reading-frame.
  • the UPR-CYC1p-lacz constructs (pCS2-1 to 5) are integrated into the LEU2 locus of BstEII cleaved strain W3116 after linearization of the plasmids with BstEII. Correct gene integration and gene replacement events are verified by PCR. Subsequently, the amplified fragments are analyzed by agarose gel electrophoresis.
  • Example 4 Protein malfolding in the endoplasmic reticulum. caused bv the olvcosylation inhibitor tunicamvcin. induces UPR-CYC1p-lacz genes
  • yeast strains in Table 1 are grown as a pre-culture in SD medium (0.67% yeast nitrogen base without amino acids, 2% glucose) for 24 h at 30°C. An aliquot of the pre-culture (1%) is inoculated in YPD medium (1% bacto-yeast extract, 2% bacto-peptone, 2% glucose) and the cells are grown for 16 h at 30°C (control). For induction of protein malfolding, an aliquot of mid-logarithmic phase cultures are treated with tunicamycin (10 ⁇ g/ ml; Sigma) for 6 h at 30°C. The cells are harvested and washed with 0.9% NaCI.
  • ⁇ -galactosidase activity measured as o-nitrophenyl- ⁇ -D-galacto-pyranoside hydrolysis at 420 nm, is normalized to cell culture density and expressed as arbitrary units.
  • the results, which are an average of three individual integrants (each assay having been performed in duplicate) are depicted in Table 2. TABLE2. Induction of ⁇ -galactosidase by tunicamycin in yeast strains harboring UPR-lacz
  • Example 5 Construction of a 2-micron plasmid which encodes an expression cassette for wild type CPY
  • the plasmid pLV9 contains the complete PRC1 gene which encodes the yeast carboxypeptidase Y enzyme (CPY) is constructed as described in Vails et al., Cell (1987), 48, 887-897.
  • the Clal-Hindlll PRC1 fragment (containing the promoter, the coding sequence for preproCPY and the transcription terminator) from pLV9 is isolated and converted to a Sall-Sacl fragment (5' to 3') for convenient cloning in the 2-micron-based vector pDP34 (DSM 4473) and the centromere vector pFL38 (ATCC 77203).
  • the manipulations of restriction sites at the 5' and 3' ends of PRC1 are made using Sail and Sacl linkers available from Boehringer.
  • the plasmid pDP34 is an E. coli-S. cerevisiae shuttle vector, which contains the complete S. cerevisiae 2-micron plasmid and encodes the S. cerevisiae URA3 and dLEU2 genes as yeast selection markers (EP-A-340 170).
  • the plasmid pFL38 is also an E. coli-S. cerevisiae shuttle vector, which contains the centromere CEN6. an autonomously replicating sequence from S. cerevisiae (ARS) and encodes the S. cerevisiae URA3 as a yeast selection marker.
  • ARS S. cerevisiae
  • pDC13 After subcloning of the Sall-Sacl PRC1 gene fragment in pDP34 (completely digested with Sail and Sacl), one correct plasmid is named pDC13.
  • the Sall-Sacl PRC1 gene fragment is also subcloned in pFL38, which is completely digested with Sail and Sacl, to obtain pFL38/ CPY ( Figure 2).
  • the mutations incorporated in the C-terminus of the CPY polypeptide sequence are described in Table 3.
  • the site where precursor pro-CPY is processed by proteinase B to form mature CPY is between the residues Asn111 and Lys112 of the prepro-CPY sequence (shown in the one-letter code N and K in Table 3).
  • the residues which are replaced in the wild type (wt) sequence are underlined.
  • the mutations are generated by performing PCR-mediated site-directed mutagenesis on a 157 bp Munl-BamHI fragment of PRC1 as comprised in pFL38CPY (Vails et al., Cell (1987), 48, 887-897).
  • the primers used for the PCRs are depicted in Table 3.
  • a Sal-Muni fragment from pFL38/CPY (see Figure 2 and Example 5) and the mutated Munl- BamHI fragments (see above) are initially subcloned in pUC19 which is completely digested with Sail and BamHI.
  • the resultant plasmids contain a 1159 bp Sall-BamHI fragment belonging to the 5' end of PRC1. The mutations are confirmed by DNA sequencing (see Example 2).
  • Example 7 Construction of 2-micron plasmids which encode expression cassettes for mutant CPY precursor genes
  • the complete mutant genes, encoding the promoter, the coding sequence and the transcription terminator) are assembled as Sall-Sacl fragments.
  • a Sall-BamHI fragment (containing a mutated pro sequence of CPY; as subcloned in ⁇ UC19; see Example 7), a BamHI-Ncol (931 bp) and a Ncol-Sacl (546 bp) (the latter two fragments are obtained from pFL38/CPY; see Figure 1 and Example 5) are subcloned in pDP34 completely digested with Sail and BamHI.
  • the resulting plasmids are named pDC8 (encoding Mut1 PRC1) , pDC9 (encoding Mut2 PRC1) and pDC10 (encoding Mut3 PRC1). They are used for expression in yeast.
  • Example 8 The mutant PRC1 genes express malfolded proteins
  • the plasmids pDC8 (encoding Mut1 PRC1 ⁇ . pDC9 (encoding Mut2 PRC1 ⁇ . pDC10 (encoding Mut3 PRC1) and pDC13 (encoding wt PRC1) are transformed in the strain W3116-3 x lacz via electroporation (see Example 3).
  • the ⁇ -galactosidase expression induced by the mutant PRC1 genes is compared to the strain which harbors the wild type- gene (wt-gene).
  • Three individual transformants from each of the above four transformations are grown as in Example 4. Each assay is performed in duplicate. The results are depicted in Table 4.
  • the ⁇ -galactosidase activity in the strain YDC13, expressing wt PRC1. is set to the value 1.
  • Example 9 The mutated pro-CPY proteins can also be monitored as malfolded when the lacz reporter is under the control of the complete KAR2 promoter
  • an Xhol-BamHI fragment containing the CYC1 promoter is removed from the plasmid pCS2-1 and is replaced by a 630 bp Sall-BamHI KAR2 promoter fragment (Rose et al., Cell (1989), 57, 1223-1236).
  • the KAR2 promoter is isolated by PCR using the two primers with SEQ ID NO 6 and 7.
  • the template for PCR is the yeast strain S288C (ATCC 26108).
  • the resulting plasmid is named pCSFOL4. This is integrated in W3116 as described in Example 3. Correct integrations are confirmed (see Example 3).
  • One of these strains is referred to as W3116 KAR2p-lacz.
  • the plasmids pDC8 (encoding Mut1 PRCi), pDC9 (encoding Mut2 PRC1).
  • pDC10 (encoding Mut3 PRC1)
  • pDC13 (encoding wt PRC1) are transformed in the strain W3116KAR2p-lacz (see above) via electroporation (see Example 3).
  • the ⁇ -galactosidase expression induced by the mutant PRC1 genes is compared to the strain which harbors the wt gene. Three individual transformants from each of the above four transformations are grown as in Example 4. Each assay is performed in duplicate. The results are depicted in Table 5.
  • the ⁇ -galactosidase activity in the strain transformed with pDC13, which encodes wt PRC1. is set to the value 1.
  • Example 10 Construction of an expression cassette for the human ⁇ -amyloid peptide ( ⁇ A4), without any signal peptide
  • the plasmid pJC21 (encoding only ⁇ A4 with no signal sequence; expression being under the control of the GAPCL promoter) is constructed as following.
  • a Sall-Bglll fragment of the GAPCL promoter (containing a -275 bp Sall-BamHI pBR322 fragment and a -400 bp promoter fragment from the yeast GAPDH gene) is isolated by PCR using the primers with SEQ ID NO 8 and 9. Plasmid pBC1 that is constructed as described in Chaudhuri etal., Eur. J. Biochem. (1992), 206, 793-800 is used as template.
  • the double-stranded DNA encoding the 42 amino acid ⁇ A4 peptide (M ⁇ ller-Hill & Beyreuther, Annu. Rev. Biochem. (1989), 58, 287-307) is chemically synthesized using oligomers with SEQ ID NO 10711 and is amplified by PCR using two primers (SEQ ID NO 12 and 13).
  • the Bglll-EcoRI fragment of ⁇ A4 is subcloned in pUC19Bgl (the BamHI site of pUC19 has been modified to a Bglll site to form pUC19Bgl; the BamHI site is flushed with Klenow polymerase; Bglll linkers /Boehringer are ligated, which is followed by digestion with Bglll and religation) and the sequence is confirmed as in Example 2.
  • Example 11 Construction of expression cassettes for the human ⁇ -amyloid peptide ( ⁇ A4), with a signal peptide pJC22 (encoding the SUC2 signal sequence linked to ⁇ A4; expression being under the control of the GAPCL promoter), pJC26 (encoding the PRC1 prepro-sequence linked to ⁇ A4; expression being under the control of the PRC1 promoter) and pDP34-NLS- ⁇ A4 (encoding the nuclear localization sequence, NLS, from the SV40 large T antigen sequence and ⁇ A4; expression being under the control of the GAPCL promoter) are constructed in a way similar to the construction of pJC21 (see Example 10; Table 6).
  • a Sall-Bglll fragment containing the GAPCL promoter linked to the SUC2 signal sequence (i. e. pBR322-GAPCLp-lnvss) is amplified by PCR using the primers with SEQ ID NO 8 and 16. Plasmid pBC1 (see Example 10) is used as template.
  • a Sall-Bglll fragment containing the PRC1 promoter linked to the prepro sequence of CPY is amplified by PCR using the primers with SEQ ID NO 17 and 18, the template being pLV9 (see Example 5 and 6).
  • a Sall-Bglll fragment (i. e. pBR322-GAPCLp-NLS) containing the GAPCL promoter linked to the nuclear localization sequence from SV40 T antigen is amplified by PCR using the primers with SEQ ID NO 8 and 19.
  • the template for the PCR is the plasmid pRH3 which encodes the -275 bp Sall-BamHI fragment from pBR322, a -400 bp BamHI-EcoRI GAPCLp fragment and an EcoRI-Spel fragment of the nuclear localization sequence from SV40 T antigen.
  • This nuclear localization sequence (Nelson & Silver, Mol. Cell. Biol. (1989) 9, 384-389) has been chemically synthesized using the deoxyoligoribonucleotide with SEQ ID NO 30.
  • Example 12 Yeast transformation of PJC21. pJC22, pJC26 and PDP34-NLS- BA4 and ⁇ - galactosidase assay of transformants
  • the plasmids are transformed in the strain W3116-3 x lacz (see Example 3).
  • ⁇ - galactosidase activity is measured (as in Example 4), see Table 7.
  • Example 13 Construction of C-terminal truncations of ⁇ A4; yeast transformation; ⁇ - oalactosidase assay
  • Two plasmids pJC24 (encoding the SUC2 signal sequence and a truncated version of ⁇ A4, 1-39) and pJC25 (encoding the SUC2 signal sequence and a truncated version of ⁇ A4, 1; 3fi) are constructed as described below. Both expression cassettes are under the control of the GAPCL promoter.
  • the peptides ⁇ A4, 1-39 and ⁇ A4, 1-36 have slower rates of aggregation in vitro (Burdick et al., J. Biol. Chem. (1992), 267, 546-554; Barrow et al., Mol. Biol. (1992), 225, 1075-1093; C. Pike etal., J. Neuroscience (1993), 13, 1676-1687).
  • Two Sall-Xbal fragments pBR322-GAPCLp-lnvss- ⁇ A4 1 ⁇ and pBR322-GAPCLp-lnvss- ⁇ A4.1-36 are isolated by PCR using the two primers (SEQ ID NO 8 and 20 for the former and SEQ ID NOs 8 and 21 for the latter construct).
  • the template is pJC22.
  • An Xbal-Sacl terminator fragment from the yeast SUC2 gene is amplified by PCR using yeast genomic DNA (from the wild type strain S288C) as template.
  • the primers have SEQ ID NOs 22 and 15.
  • the Sall-Xbal fragment and an Xbal-Sacl fragment is subcloned in pDP34 completely digested with Sail and Sacl (see Table 8).
  • the plasmids are transformed in the strain W3116-3 x lacz (see Example 3).
  • ⁇ - galactosidase activity is measured (as in Example 4) and compared with the strains YJC21 and YJC22, described in Example 10 (see Table 9).
  • Example 14 Construction of the rat mutant ⁇ A4 (mr ⁇ A4) and the inverted human ⁇ A4 (ifiA41
  • pJC27 and pJC28 are constructed as shown in Table 10. Both expression cassettes contain the prepro sequence of the PRC1 gene and are driven by the PRC1 promoter.
  • pJC27 encodes the rat mutant ⁇ A4 (mr ⁇ A4) (Dyrks et al., FEBS Lett. (1993), 324, 231-236; Hilbich et al., Mol. Biol. (1991 ), 218, 149-163) and pJC28 encodes the inverted human ⁇ A4 (i ⁇ A4) (Fraser et al., Biochemistry (1992), 31 , 10716-10723).
  • the plasmids are constructed of a Sall-Bglll fragment (containing the CPY promoter and the prepro sequence of CPY), a Bglll-EcoRI fragment (containing either mr ⁇ A4 or i ⁇ A4) and an EcoRI-Sacl fragment of the SUC2 terminator.
  • the Bglll-EcoRI fragments (containing either mr ⁇ A4 or i ⁇ A4) are synthesized using two overlapping oligodeoxyribonucleotides (see SEQ ID NOs 25/26 and 27/28) and two primers (see SEQ ID NOs 12 and 13), which hybridize to the 5' and 3' end of the fragments.
  • Sall-Bglll, the Bglll-EcoRI and the EcoRI-Sacl fragment is subcloned in pDP34 completely digested with Sail and Sacl.
  • amino acid sequences of ⁇ A4, mr ⁇ A4 and i ⁇ A4 are shown in Table 11. The differences from the wt human ⁇ A4 sequence are underlined.
  • the plasmids in Table 10 are transformed in the strain W3116-3 x lacz (see Example 3). ⁇ - galactosidase activity is measured as in Example 4 and compared with the strains YJC26, YJC21 and YJC22, described in Example 10 (see Table 12).
  • Example 15 Trivalent aluminum and divalent zinc increase malfolding of ⁇ A4 in yeast
  • ⁇ -galactosidase activity is measured as in Example 4 after cells are grown in YPD in the presence of AICI 3 (-3 mM; Merck) and ZnCI 2 ( ⁇ 300 ⁇ M; Merck). The results are described in Table 13.
  • ⁇ -GA represents the ⁇ -galactosidase activity.
  • the symbol "-ive control" implies that the cells have been grown in the absence of either of the two transition metal cations.
  • Example 16 Desferal and ascorbic acid reduce malfolding of ⁇ A4 ⁇ A4 is expressed in the minimal medium SD (see Example 4) in the absence or presence of desferrioxamine (Desferal ® CIBA-GEIGY) and ascorbic acid (Merck), ⁇ -galactosidase activity is measured as in Example 4. The results are described in Tables 14 and 15. ⁇ -GA represents the ⁇ -galactosidase activity. The symbol "-ive control" implies that the cells have been grown in the absence of either desferrioxamine or ascorbic acid.
  • microorganism strains were deposited at the Deutsche Sammlung von Mikroorganismen (DSM), Mascheroder Weg 1 b, D-38124 Braunschweig (accession numbers and deposition dates given):
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (gen r ⁇ c)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (gencmic) (ix) FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AIXACAACTG CITCGAAAGA AGCAAAACTT CGTTGTGGTG CAACTTATGC CAAGACTGGT 60

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Abstract

L'invention concerne un nouveau procédé de détection de protéines mal repliées, ainsi que des procédés de mesure de la quantité de protéines mal repliées produite et de détection des composés influençant cette structure.
PCT/EP1995/003475 1994-09-16 1995-09-04 Procede de detection de proteines mal repliees WO1996008561A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU35215/95A AU3521595A (en) 1994-09-16 1995-09-04 Method for detection of malfolded protein
EP95931986A EP0783574A1 (fr) 1994-09-16 1995-09-04 Procede de detection de proteines mal repliees
JP8509869A JPH10506271A (ja) 1994-09-16 1995-09-04 異常折りたたみ蛋白質を検出するための方法
FI971034A FI971034L (fi) 1994-09-16 1995-09-04 Menetelmä väärinlaskostuneen proteiinin toteuttamiseksi
MXPA/A/1997/001881A MXPA97001881A (en) 1994-09-16 1997-03-12 Method for the detection of protein bad pleg

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EP94810536.6 1994-09-16
EP94810536 1994-09-16

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WO2001044816A3 (fr) * 1999-12-15 2002-05-30 Univ California Regulation conformationnelle et topologique des proteines
WO2005038024A1 (fr) * 2003-10-16 2005-04-28 Novozymes A/S Methode de criblage destinee a identifier une proteine secretant des cellules hotes recombinantes

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001044816A3 (fr) * 1999-12-15 2002-05-30 Univ California Regulation conformationnelle et topologique des proteines
US6821742B2 (en) 1999-12-15 2004-11-23 Regents Of The University Of California Conformational and topological protein regulation
US7041462B2 (en) 1999-12-15 2006-05-09 Regents Of The University Of California Conformational and topological protein regulation
WO2005038024A1 (fr) * 2003-10-16 2005-04-28 Novozymes A/S Methode de criblage destinee a identifier une proteine secretant des cellules hotes recombinantes
US8846403B2 (en) 2003-10-16 2014-09-30 Novozymes A/S Method of screening for protein secretion recombinant host cells

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