WO1991016436A1 - Nouveaux dosages de proteases - Google Patents

Nouveaux dosages de proteases Download PDF

Info

Publication number
WO1991016436A1
WO1991016436A1 PCT/US1991/002283 US9102283W WO9116436A1 WO 1991016436 A1 WO1991016436 A1 WO 1991016436A1 US 9102283 W US9102283 W US 9102283W WO 9116436 A1 WO9116436 A1 WO 9116436A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
protease
regulatory protein
peptide
hybrid regulatory
Prior art date
Application number
PCT/US1991/002283
Other languages
English (en)
Inventor
Bimalendu Dasmahapatra
Original Assignee
Schering Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Schering Corporation filed Critical Schering Corporation
Publication of WO1991016436A1 publication Critical patent/WO1991016436A1/fr

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding

Definitions

  • proteases play critical roles in a number of important human and animal diseases. In some cases, proteases are essential for the replication of microbial pathogens. In other cases they may have harmful effects on target tissues and organs independent of any microbial involvement.
  • picornaviruses which are currently divided into four genera: Enterovirus. Cardiovirus. Rhinovirus and Aphthovirus (foot-and-mouth disease viruses). Coxsackieviruses are picornaviruses within the Enterovirus group which cause a wide spectrum of human diseases, ranging from mild respiratory illness to more serious disseminated diseases of the nervous system and heart (Melnick, J.L., in Virology, 1985, B.N. Fields, Ed., Raven Press, New York, pp. 739- 794).
  • the genomes of all picornaviruses comprise a messenger- active single-stranded RNA molecule.
  • This RNA encodes a single large polyprotein that is cleaved to produce both structural and non-structural mature viral proteins.
  • these cleavages are carried out by two virally-encoded proteases designated 2A and 3C.
  • the 2A protease is responsible for the primary cleavage that generates the viral capsid precursor P1 [Toyoda et al., Cell 45:761 (1986)].
  • the 3C protease which is itself released from the polyprotein by an autocatalytic cleavage, then carries out all but one of the remaining cleavage reactions [Hanecak et al., Cell 2Z:1063 (1984)]. This protease specifically cleaves the peptide bond between certain adjacent glutamine and glycine residues.
  • HIV-1 human immunodeficiency virus type 1
  • the HIV-1 protease which is part of the ⁇ l gene product and is instrumental in the cleavage of large precursor proteins into structural and functional viral proteins, is specific for HIV proteins [Ratner et al., Nature 313:277 (1985); Farmerie et al., Science 2S£:305 (1987)].
  • proteases are involved in the destructive effects of microbial pathogens in ways that do not involve replicative processes.
  • Clostridium histolvticum a bacterium that causes gas gangrene, secretes a battery of collagenases that produce multiple cleavage of collagen.
  • These collagenases which specifically cleave at the amino side of glycine residues in peptide linkages, contribute to the invasiveness of the bacterium by destroying the connective-tissue barriers of the host.
  • the bacterium itself is unaffected by its secretory products because it, unlike the multicellular eukaryotes it infects, is devoid of collagen.
  • This invention provides hybrid regulatory proteins comprising a DNA-binding peptide, a linker peptide susceptible to cleavage by a specific protease, and a DNA transcription-modifying peptide; which hybrid proteins are rendered substantially inactive by cleavage of the linker peptide by the protease.
  • DNAs encoding such hybrid proteins and recombinant vectors comprising such DNAs.
  • These recombinant vectors can be inserted into eukaryotic host cells, thereby forming the basis of relatively rapid and specific assays for proteolytic enzymes of medical and biochemical interest.
  • Eukaryotic cells transformed with such recombinant vectors are also part of this invention.
  • This invention further provides methods for identifying inhibitors of specific proteases, comprising:
  • a DNA encoding a hybrid regulatory protein comprising a DNA-binding peptide, a linker peptide susceptible to cleavage by a specific protease, and a DNA transcription- modifying peptide, which hybrid regulatory protein is rendered substantially inactive by cleavage of the linker peptide by the protease;
  • an inhibitor of the specific protease is identified by measuring the rate of production of the indicator gene product and comparing such rate to the rate of production of the indicator gene product in the absence of the inhibitor.
  • FIG. 1 is a schematic representation of the construction of plasmids p1009 and p1013.
  • hybrid regulatory protein means a protein comprising three peptides (polypeptides) joined in covalent peptide linkage which together can (a) bind to a specific region of DNA that controls expression of one or more genes or cDNAs and (b) by thus binding alter the rate of transcription of the genes or cDNAs.
  • the alteration in the rate of transcription can be either positive (i.e., the hybrid protein may be a transcription activator) or negative (i.e., the protein may act as a repressor).
  • Some of the hybrid regulatory proteins of the invention are derivatives of the yeast transcription activator GAL4, a protein of 881 amino acids that is required for galactose catabolism in the yeast £. cerevisiae. Ma et al. [Cell 4£:847 (1987)] have reported that GAL4 contains two distinct functional domains - a DNA-binding domain containing amino acid residues 1-147 and two activating domains. One activating domain consists of amino acid residues 148-236, the other, residues 768-881. A construct containing the DNA-binding domain and either of the activating domains will function as a DNA transcriptional activator. Therefore, a large in-frame deletion of about the middle third of GAL4 (deleting the first activating domain but retaining the second) does not abolish DNA transcriptional activator activity [Ma et al., Cell 4£:847 (1987)].
  • a fragment of GAL4 consisting of only residues 1-147 retains the ability to bind DNA but cannot activate transcription.
  • the addition of some random acidic peptides containing as few as 15 amino acid residues to the carboxyl terminus of the 1-147 fragment can confer transcription activating activity on the fragment [Giniger et al., Nature 22Q:670 (1987)].
  • the GAL4-based hybrid regulatory proteins of the invention comprise:
  • linker peptide susceptible to cleavage by a specific protease, which linker peptide contains from about 10 to about 250 amino acid residues;
  • the DNA-binding peptide was to the left of the linker peptide and the transcription-activating peptide was to the right, as viewed from the amino to the carboxyl terminus, the positions of the elements flanking the linker peptide could as well have been reversed.
  • the only requirement for any of the hybrid regulatory proteins of the invention is that the DNA-binding peptide and the transcription-modifying peptide be bridged by the linker peptide.
  • the linker peptides used in the construction of the hybrid protein can be susceptible to cleavage by specific proteases acting in the cis or trans configuration.
  • proteases acting in the cj ⁇ configuration themselves comprise the linker peptide.
  • These proteases are transcribed and translated from the DNAs encoding the hybrid proteins, and they are autoproteolytic. As a result of their activity, the DNA- binding and transcription-activating regions of the hybrid proteins are separated, rendering the cleaved hybrid proteins substantially inactive compared to the intact proteins.
  • substantially inactive means that a hybrid regulatory protein cleaved by a protease has less than about 20%, preferably less than about 10% and most preferably less than about 5% of the transcription-modifying activity of the uncleaved, intact protein.
  • protease of interest itself serves as the linker peptide, it is desirable to place additional amino acid residues at the amino and/or carboxyl termini of the protease. Such additional residues provide flexibility to permit the protease to assume an active
  • residues can be encoded, e.g.. by flanking regions of DNA (a gene or cDNA) encoding the protease.
  • nucleic acid sequences coding for other residues can be engineered in frame into the DNA encoding the protein, using known methods.
  • additional amino acid residues are added to the end(s) of the protease.
  • about 30 additional residues flank both the amino and carboxyl termini of a Coxsackievirus 3C protease linker peptide.
  • about 20 to 25 additional residues flank both the carboxyl and amino termini of an HIV-1 protease linker peptide.
  • the linker peptide is not a protease itself but has an amino acid sequence which is recognized and specifically cleaved by the protease.
  • the linker peptide should contain regions of the viral protein known to be susceptible to cleavage by the protease (Toyoda et al., supra: Hanecak et al., supra).
  • HIV-1 protease and collagenase require linker peptides having amino acid sequences corresponding to the sequences of their well-known substrates [Meek et al., Proc. Natl. Acad. Sci. USA fi£:1841 (1989) (HIV protease); Miller et al., Arch. Biochem. Biophys. 139:104 (1970) (collagenase)]. Trypsin-like proteases specifically cleave at the carboxyl side of lysine and arginine residues. Chymotrypsin-like proteases are specific for cleavage at tyrosine, phenylalanine and tryptophan residues, etc.
  • proteases mentioned above are only some of the enzymes that can be assayed by the methods of this invention, and the invention encompases any protease for which the gene (or cDNA) and substrate specificity are known or determinable.
  • the linker peptide will contain two or more repeats of the susceptible residues, to maximize the probability of cleavage within the linker peptide.
  • the natural substrate of a given protease can be used as the linker peptide.
  • a linker peptide having an amino acid sequence corresponding to that of a collagen molecule can be used, when the protease is a collagenase.
  • GAL4-based hybrid regulatory proteins are illustrative of activator-type hybrids that can be made according to the present invention.
  • Other eukaryotic or prokaryotic transcription activators can be modified in analogous fashion. All that is required is that an activator selected have discernible DNA-binding and transcription-activating regions or domains that can be covalently joined by a suitable protease-sensitive linker peptide.
  • Hybrid transcription repressor proteins are also encompassed by this invention.
  • the phage ⁇ repressor can be modified for use in a protease assay system.
  • the ⁇ repressor which is encoded by the cl gene, contains two functional domains.
  • An amino-terminal domain consists of amino acid residues 1-92, using the numbering of the amino acid sequence disclosed by Sauer [Nature 2_Z£:301 (1978)]. This domain binds to specific operator (promoter) sites in DNA.
  • a carboxyl-terminal domain consisting of residues 132-236 is instrumental in causing dimerization of the repressor. Dimerization greatly enhances repressor activity.
  • the isolated amino-terminal domain can bind DNA specifically, but it dimerizes very poorly and as a result has an operator affinity that is several orders of magnitude lower, compared to that of the intact protein [Johnson et al., Proc. Natl. Acad. Sci. USA Z£:5061 (1979)].
  • Hybrid regulatory proteins of this invention based upon the ⁇ repressor comprise:
  • linker peptide susceptible to cleavage by a specific protease, which linker peptide contains from about 10 to about 250 amino acid residues
  • a DNA transcription-modifying peptide having an amino acid sequence corresponding to the sequence of about the last 105 residues of the carboxyl terminus of the phage ⁇ repressor.
  • the foregoing three peptides are joined end-to-end in peptide linkage to form the complete hybrid regulatory protein.
  • the amino acid residue numbers mentioned above relate to the complete amino acid sequence of the ⁇ repressor disclosed by Sauer, supra.
  • linker peptides used in the construction of such hybrid repressor proteins are as described above for hybrid transcription activator proteins.
  • the invention is not limited to ⁇ repressor-based hybrid proteins but also encompasses proteins based upon any repressor comprising (a) a specific DNA binding domain and (b) a second domain, the presence of which is required for the effective functioning of the repressor.
  • DNAs encoding the hybrid regulatory proteins can be chemically synthesized. Such synthesis can be carried out using any of the known methods, such as the phosphoramidite solid support method of Matteucci et al. [J. Am. Chem. Soc.102:3185 (1981 )].
  • cDNA can be made from messenger RNA (mRNA) isolated from cells known to make the starting regulatory protein, protease and/or protease-susceptible linker peptide, using standard techniques.
  • mRNA messenger RNA
  • protease and/or protease-susceptible linker peptide can be used to produce double-stranded cDNA a* described, e.g.. by Maniatis et al., supra.
  • This cDNA can be inserted into an appropriate cloning vector which can be used to transform E. coli. to produce a cDNA library.
  • the cDNA library can be screened using oligonucleotide probes based upon the known nucleotide sequences of DNAs encoding the desired starting regulatory protein, protease and or linker peptide.
  • probes can be radiolabeled, e.g.. by nick-translation using Pol I DNA poiymerase in the presence of the four deoxyribonucleotides, one of which contains 32 P in the ⁇ position (Maniatis et al., ⁇ ujara, page 109). Genomic libraries can be probed in a similar fashion.
  • the elements needed to construct the hybrid regulatory protein-encoding DNAs of the invention are cleaved by restriction endonucleases as required and inserted into an appropriate eukaryotic cell expression vector.
  • Plasmid pDSVS contains a ⁇ -lactamase gene (conferring ampicillin resistance), a pBR322 origin of replication, a dihydrofolate reductase (DHFR) transcription cassette, an SV-40 promoter, a polylinker containing a number of unique restriction sites, and an SV-40 late region polyadenylation signal sequence.
  • DHFR dihydrofolate reductase
  • Plasmid pDSRS is similar to pDSVS except for the substitution of an SR ⁇ promoter for the SV-40 promoter of pDSVS.
  • Plasmid pSRS is similar to pDSRS, except that the DHFR transcription cassette has been deleted in pSRS.
  • Plasmids pDSRG and pSRG are similar to pDSRS and pSRS, respectively, except that pDSRG and pSRG contain a human ⁇ -globin polyadenylation signal sequence instead of an SV-40 late region polyadenylation signal sequence.
  • Plasmids pDSVS, pDSRS, pDSRG, pSRS and pSRG were deposited February 21, 1990 with the American Type Culture Collection, Rockville, MD, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures. They have been assigned Accession Nos. ATCC 68231, 68232, 68233, 68234 and 68235, respectively.
  • a vector selected will be one that can also be cloned in a bacterium such as £• coli. to facilitate recombinant vector construction and replication.
  • Any vector having control elements operable in yeast cells can also be used in this invention.
  • Methods for preparing useful yeast vectors and strains are well known in the art and have been described, e.g.. by Ma et al., Gene 5&201 (1987).
  • Insertion of the DNA into a cloning and expression vector is easily accomplished when both the DNA and the vector have been cut with the same restriction enzyme(s), since complementary DNA termini are thereby produced. If this cannot be accomplished, it may be necessary to modify the cut ends that are produced by digesting back single-standard DNA to produce blunt ends, or by achieving the same result by filling in the single-stranded termini with an appropriate DNA poiymerase. In this way, blunt-end ligation with an enzyme such as T4 DNA ligase may be carried out.
  • any site desired may be produced by ligating oligonucieotides (linkers) onto the DNA termini. Such linkers may comprise specific oligonucleotide sequences that encode restriction site recognition sequences.
  • the cleaved vector and the isolated DNAs may also be modified by homopolymeric tailing, as described by Morrow [Meth. Enzymol. fifi:3 (1979)].
  • Many of the cloning and expression vectors that can be used in this invention contain one or more marker activities that can be used to select for desired transformants, such as ampicillin or tetracycline resistance. Selection of host cells into which such vectors have been inserted is greatly simplified when the host cells otherwise lack the activities contributed by the vectors.
  • nucleotide sequences of DNAs inserted at a selected site in a cloning vehicle may include nucleotides that are not part of the actual structural genes.
  • additional nucleotides needed to place the inserted DNAs in frame with translation initiation signals may be present, or linkers may be ligated to the DNAs to adapt them to the cut ends of the vehicle for insertion.
  • a DNA sequence encoding a linker peptide which itself is a protease should preferably contain nucleotides encoding additional amino acid residues, so that the protease will be able to achieve an active conformation.
  • DNAs encoding the hybrid regulatory proteins of the invention are under the control of a regulatable promoter, so that expression of the DNAs can be regulated by standard methods until test compounds are introduced into the assay systems.
  • a regulatable promoter e.g.. the ⁇ PR promoter under the control of the ⁇ cP gene
  • chemical activation or repression e.g.. the lacZ promoter under the control of isopropyl- ⁇ -d- thiogalactopyranoside.
  • expression of a gene encoding a hybrid transcription activator under the control of the ADHI promoter [Bennetzen et al., J. Biol. Chem.
  • 25Z:3018 (1982)] was induced by growth in a galactose and glycerol-containing medium.
  • the recombinant vectors of the invention encoding the hybrid regulatory proteins are used in conjunction with a responsive site that is operatively linked to an indicator gene.
  • DNA bearing a responsive site and linked indicator gene can be introduced into a host cell in a vector. Alternatively, it can be integrated into a chromosome of the cell, using standard methods for stable cell transformation.
  • DNA site responsive to a hybrid regulatory protein means a DNA site to which the hybrid regulatory proteins specifically bind and thereby modify (either increase or decrease) transcription of an operatively linked indicator gene.
  • indicator gene means a gene or cDNA which encodes an expression product that can readily be measured.
  • UASG for Upstream Activating Sequence of Galactose
  • yeast Saccharomyces cerevisiae GAL4 recognizes four sites in UASG and, by binding to those sites, upregulates galactose catabolism. Complete UASG. the nucleotide sequence of which has been disclosed by Guarente et al. [Proc. Natl. Acad. Sci. USA 7J-.:7410 (1982)] and Johnston et al. [Mol. Cell. Biol. 4:1440 (1984)], can be used in yeast cells.
  • UASG is a natural yeast cis-activating sequence
  • GAL4-binding element thereof can also be used in mammalian cells.
  • Kakidani et al. [Cell 52:161 (1988)] have reported that a plasmid bearing UASG (or a 17-mer element of it) upstream of the start site of the MMTV promoter responded to GAL4 in Chinese hamster ovary (CHO) cells by stimulating transcription of a chloramphenicol acetyltransferase (CAT) gene.
  • CAT chloramphenicol acetyltransferase
  • UASQ or an element thereof employed in conjunction with the MMTV promoter in mammalian cells as described by Kakidani et al. can thus be used in this invention.
  • Lambda repressor-based hybrid regulatory proteins can be used in conjunction with the P ⁇ _ or PR promoter [Rosenberg et al., Meth. Enzymol. 101:123 (1983)].
  • reporter genes can be operatively linked to one of the foregoing DNA fragments.
  • suitable reporter genes include but are not limited to £. coli ⁇ -galactosidase [An et al., Mol. Cell. Biol. 2:1628 (1982)], xanthine- guanine phosphoribosyl transferase [Chu et al., Nucleic Acids Res. 12:2921 (1985)], galactokinase [Shumperli et al., Proc. Natl. Acad. Sci.
  • Expression products of the reporter genes can be measured using standard methods. For example, bioassays can be carried out for biologically active proteins such as interleukin-2. Enzyme assays can be performed when the reporter gene product is an enzyme such as alkaline phosphatase. Alternatively, various types of immunoassays such as competitive immunoassays, direct immunoassays and indirect immunoassays may be used.
  • label includes moieties that can be detected directly, such as fluorochromes and radiolabels, and moieties such as enzymes that must be reacted or derivatized to be detected.
  • samples from induced cultures are incubated with an antibody against the reporter gene product and a known amount of labeled reporter gene product. Any unlabeled product produced by the cells competes with the labeled material for binding to the antibody. The resulting immune complexes are separated and the amount of labeled complex is determined.
  • the reporter gene product produced by the cells can be quantified by comparing observed measurements to results obtained from standard curves.
  • Direct immunoassays involve incubating culture samples with a labeled antibody against the reporter gene product and separating any immune complexes that form. The amount of label in the complexes is determined and can be quantified by comparison to standard curves.
  • Enzyme-linked immunosorbant assays can also be carried out by well known methods, e.g., as described in U.S. patent No. 4,665,018 to Void.
  • label used will depend upon the type of immunoassay used.
  • labels include, e.g., radiolabels such as 32 P, 125 ⁇ 3H anc j 14 ; fluorescent labels such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone; chemiluminescers such as luciferia and 2,3-dihydrophthal-azinediones; and enzymes such as horseradish peroxidase, alkaline phosphatase, lysozyme and glucose-6-phosphate dehydrogenase.
  • the antibody or reporter gene product can be tagged with such labels by known methods.
  • coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bisdiazotized benzadine and the like may be used to tag the antibodies with fluorescent, chemiluminescent or enzyme labels.
  • Host mammalian cells that have been used to express various recombinant proteins and which can be used in this invention include but are not limited to the African green monkey kidney (COS), Chinese hamster ovary (CHO), NS-1 , SP2/0, NIH 3T3, NIH 3T6, C127, CV-1 , HeLa, mouse L and Bowes cell lines. Any of the known yeast cell lines can also be used. Cells selected should preferably not normally produce the kind of regulatory protein employed. Alternatively, the cells can be engineered by standard methods to eliminate endogenous synthesis of the protein.
  • GAL4-based regulatory proteins can be used in unaltered mammalian cells, since GAL4 is a natural yeast transcription activator.
  • Use of GAL4-based hybrid proteins in yeast cells is preferably carried out after prior elimination of endogenous GAL4 production in the cells. This can be accomplished using known methods, such as those described by Ma et al. [Cell 45:847 (1987)] or Rothstein [Meth. Enzymol. 101 :202 (1983)]. Such methods result in replacement of the GAL4 gene with a selectable marker such as URA3 or LEU2.
  • a yeast strain deleted for both GAL4 and GAL80, a negative regulator of GAL4 was used, although the latter deletion was not necessary for use in this invention.
  • a recombinant plasmid encoding a hybrid regulatory protein is introduced into a host cell containing a responsive site/indicator gene. If the protease to be assayed does not itself comprise the protease-sensitive linker peptide of the protein, a second recombinant plasmid encoding the protease is introduced into the cell. Finally, the cell is contacted with a potential inhibitor of the protease, expression of the regulatory protein is induced, and the rate of production of the indicator gene product is measured.
  • Inhibition of the protease in an assay employing a repressor-based hybrid regulatory protein will be detected by a decrease in the measured rate of expression of the indicator gene product, compared to the rate in the absence of the inhibitor. The opposite effect will be observed in an activator-based assay. In either case, depending upon the inhibitory power and concentration of the inhibitor, the rate of indicator gene expression will change and will, if inhibition of the protease is complete, reach the level produced by the uncleaved, intact hybrid regulatory protein.
  • a control test of the compound in parallel. This is done by exposing control host cells of the same type which harbor an indicator gene not under the control of the hybrid regulatory protein to the compound and measuring the rate of production of this indicator gene product, compared to the rate of production in the absence of the compound.
  • a specific inhibitor of the protease will alter the rate of production of the indicator gene product in the cells in which the indicator gene is under the control of the hybrid protein, but not in the control cells.
  • Such control tests are generally unnecessary, however, when the hybrid regulatory protein is a transcription activator.
  • E.. j£li strain XL1-Blue (Stratagene Inc., La Jolla, CA) was used for cloning all plasmid constructs.
  • the bacteria were grown at 37 °C in LB broth (Maniatis et al., supra, page 68) containing 100 ⁇ g/ml ampicillin.
  • Yeast strain GGY1 ::171 ( ⁇ GAL4, ⁇ GAL80, Ura3, His 3 and Leu 2) carries a GAL1-lacZ fusion gene activated by GAL4 at the UASG site and does not produce its own GAL4. Stimulation of this and similar strains by exogenous GAL4 produces a readily measured increase in ⁇ -galactosidase activity.
  • yeast cells were grown in YEPD medium [1% yeast extract (DIFCO Laboratories, Detroit, Ml), 2% Bacto Peptone (Difco), 0.004% adenine sulfate (Sigma Chemical Co., St. Louis, MO) and 2% dextrose (Sigma); Sherman et al., Methods in Yeast Genetics. 1983, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] at 30°C to log phase (5 x 10 6 to 10 7 cells/ml). Growth was followed by measuring OD ⁇ oo in a conventional spectrophotometer, with 1 OD ⁇ oo unit equivalent to about 10 7 cells per ml.
  • YEPD medium 1% yeast extract (DIFCO Laboratories, Detroit, Ml), 2% Bacto Peptone (Difco), 0.004% adenine sulfate (Sigma Chemical Co., St. Louis, MO) and 2% dextrose (Sigma); Sherman et al., Methods in Yeast Genetics. 1983, Cold
  • the cells were washed and then cultured at 30°C in YEPGal-Gly medium containing 1% yeast extract, 2% peptone, 0.004% adenine sulfate, 2% galactose and 2% glycerol. Incubation in this medium (induction medium) was carried out for about 20 hours prior to assay.
  • Liquid ⁇ -galactosidase assays were performed by aliquoting 50 ⁇ l of yeast cells in induction medium into 96-well microtiter plates (Falcon) at a concentration of 1 x 10 6 cells/ml. Additional induction medium containing lithium acetate was added to each well to give a final lithium acetate concentration of 10 mM and a final volume of 250 ⁇ l. Inclusion of the lithium acetate in the medium facilitated the uptake of small molecules by the yeast cells. The plates were incubated at 30°C for about 20 hours, after which the cells were pelleted by centrifuging the plate at 4,000 x g for 30 minutes, and the supernatant fluid was removed from each well by gentle aspiration.
  • ⁇ -galactosidase activity was measured by adding 100 ⁇ l of freshly-prepared MUG Reagent [Geballe et al., Cell 45:865 (1986); Mallon et al., J. Virol.54:6282 (1990)] mixture [4:1 (vol:vol) MUG Reagent (12.5 mg 4-methylumbelliferyl ⁇ -D-galactoside in 100 ml distilled water): buffer (0.6 M Na 2 HP0 4 , 0.4 M NaH 2 P0 4 , 0.01 M KCI, 0.001 M MgS ⁇ 4, 2.7 ml/liter ⁇ -mercaptoethanol)] to each well. The plates were incubated at 37°C for one to two hours and then read on a Dynatech (Chantilly, VA) Microfluor plate reader with the excitation and emission wavelengths set at 365 and 450 nm, respectively.
  • £. coli strain XL1-Blue was transformed using CaCl2 as described by Maniatis et al., supra, page 250. Transformants were selected on plates containing 100 ⁇ g/ml ampicillin.
  • Yeast strain GGY1 ::171 was transformed using lithium acetate as described by Ito et al., supra. Plasmids were introduced and selected on appropriate dropout plates (Sherman et al., supra).
  • plasmid pMA236 contains a truncated GAL4 gene in which nucleotides encoding residues 1-147 of the DNA-binding domain and residues 768-881 of the transcription- activating domain of GAL4 are joined by a unique EcoRI restriction site. Plasmid pMA236 was modified by cleavage at the EcoRI site and insertion of a functional (p1009) or non-functional (p1013) Coxsackievirus 3C protease at the cleavage site. Both constructions are shown schematically in Fig. 1.
  • plasmid pCB11135 [Tracy et al., Arch. Virol. 55:157 (1985)] was digested with Pstl and Aval, and a fragment containing the CVB3 sequence (including the 3C coding region) was iigated into the Estl and Aval sites of transcription plasmid pGEM2 (Promega Corp., Madison, Wl).
  • the resulting plasmid pB1C13 was digested with Xbal and treated with the Klenow fragment of DNA poiymerase I to fill in the 5' protruding ends.
  • Plasmid p1009 was constructed by cleaving pC1 B1 with bal and BamHl and isolating an 1100 bp fragment encoding wild-type Coxsackievirus 3C protease. The isolated fragment was further digested with Hinfl and treated with Klenow fragment to fill in the cut ends. A 795 bp DNA fragment was then isolated and iigated to plasmid pMA236 which had been deaved with EcoRI. treated with calf intestinal phosphatase to remove 5' phosphate groups and end-filled with Klenow fragment. Plasmid p1009 contains nudeotides corresponding to bases 5254 to 6052 of the CVB3 gene sequence.
  • plasmid pC11 B9 was made by inserting a 12 bp linker (GGATCCGGATCC) at the Ball site of plasmid pC1B1.
  • This in-frame insertion which encoded an additional four amino acid residues on the carboxyl side of residue 166 of the wild-type Coxsackievirus 3C protease, produced a protein that was proteolytically inactive in both the ej ⁇ and trans configurations.
  • Plasmid p1013 was constructed by cleaving pC11B9 with Xbal and fiamHI, and isolating an 1100 bp fragment. This fragment was digested with Hinfl and blunt ended with Klenow fragment, and an 807 bp fragment was isolated. This isolated fragment was Iigated to pMA236 which had been linearized with EcoRI. dephosphorylated and Klenow blunt ended.
  • Both plasmid p1009 and p1013 were characterized by restriction enzyme analysis and by nucleotide sequencing [primer extension method; Sanger et al., Proc. Natl. Acad. Sci. USA 24:5463 (1977)] at the junction of the yeast and viral sequences.
  • Plasmids p1009 and p1013 were transformed into yeast strain
  • the transformants were then streaked onto an X-gal plate, cultured and induced with galactose and glycerol as described above.
  • Transformants containing plasmid pMA210 were also prepared and cultured on the plate, together with untransformed strain GGY1 ::171 and the p1009 and p1013 transformants.
  • Plasmid pMA210 [Ma, Cell 45:847 (1987)] encodes the full wild-type GAL4 protein.
  • Yeast cells harboring plasmids p1013 and pMA210 produced appreciable amounts of ⁇ -galactosidase activity, as evidenced by visible blue spots on the plate at the positions of the cells. Both of these cells produced intact transcription activators which bound to the UASG site, thereby turning on expression of the ⁇ -galactosidase genes operatively linked to that site.
  • untransformed yeast cells showed essentially no ⁇ -galactosidase activity (essentially no color reaction), as did cells harboring plasmid p1009.
  • the latter cells produced a hybrid regulatory protein which, due to the presence of the functional 3C protease between the DNA-binding and transcription-activating domains, underwent autoproteolysis. As a consequence, an intact transcription activator was not available in these cells to stimulate ⁇ -galactosidase production at the UASG site.
  • plasmid p1009 a plasmid designated pHP236 was constructed by inserting a DNA encoding an HIV-1 protease-linked hybrid regulatory protein into plasmid pMA236.
  • the resulting construct contained nucleotides encoding residues 1-147 of the DNA-binding domain and residues 768-881 of the transcription-activating domain of GAL4. These GAL4 elements were separated in pHP236, however, by an HIV-1 protease peptide linker.
  • plasmid pHP2366 a plasmid designated pBD1091 was first constructed by digesting ⁇ HXB-2 [a subclone of HIV-1 genomic DNA; Shaw et al., Science 225:1165 (1984)] with Clal. filling the 5' overhangs with the Klenow fragment of £. ⁇ 2li DNA poiymerase and partially digesting with sil. A 2,095 bp Clal-Nsil fragment was purified and cloned into plasmid PKK233-2 (Pharmacia LKB Biotechnology, Piscataway, NJ) which had been linearized with Ncol. blunt-ended with Klenow poiymerase and digested with Esll.
  • Plasmid pBD1091 contains DNA having the nucleotide sequence of nucleotides 829-2924 of HIV-1 [Starcich et al., Science 22Z:538 (1985); Ratner et al., Nature 212:277 (1985)] under the control of the ⁇ tc ⁇ promoter. These nucleotides encode part of gag, all of the protease and part of the reverse transcriptase of HIV-1 in a single reading frame.
  • Plasmid pBD1091 was cleaved with Bglll, blunt ended with Klenow poiymerase and Iigated to produce plasmid pBD1140, which contained a newly-created £]aj restriction endonuclease site in the HIV-1 sequence.
  • Plasmid pBD1140 was digested with Pvull and Ball, and a 1 ,509 bp DNA fragment containing the HIV-1 nucleotide sequence from nucleotides 1145-2654 was subcloned into the EcoRV site of plasmid pSP71 (Promega Corporation, Madison, Wl).
  • the resulting plasmid designated pHP71
  • pHP71 was digested with CJal and treated with Klenow poiymerase to produce a 568 bp DNA fragment containing nucleotides 2093-2654 of HIV-1 (the protease of HIV-1 is encoded by nucleotides 2286-2582) and 7 nucleotides from the vector DNA.
  • This fragment was isolated and cloned into plasmid pMA236, which had been linearized with EcoRI. treated with calf intestinal alkaline phosphatase and blunt ended with Klenow poiymerase, to produce plasmid pHP236.
  • Plasmid pHP236m an analog of plasmid pHP236 encoding a mutant, inactive HIV-1 protease linking the two functional domains of GAL4, was constructed by digesting plasmid pART2 ml 18 [Loeb et al., J. Virol. 62:111 (1989)] with figlll and Ball. A 560 bp fragment was isolated, treated with Klenow poiymerase and subcloned into pSP71 which had been digested with CJal and JtJi ⁇ dlll and blunt- ended with Klenow poiymerase. The resulting plasmid, pHP71 m, was digested with EcoRV and Xhol.
  • plasmid pHP236 and pHP236m were transformed into yeast strain GGY1 ::171. The transformants were then streaked onto an X-gal plate, cultured and induced with galactose and glycerol as described above. Untransformed strain GGY1 ::171 was also cultured on the plate together with the pHP236 and pHP236m transformants.
  • Yeast cells harboring plasmids pHP236m produced appreciable amounts of ⁇ -galactosidase activity, as evidenced by a visible blue spot on the plate at the position of the cells. Since the protease encoded by plasmid pHP236m was inactive and incapable of cleaving the linker peptide, these cells produced an intact transcription activator which bound to the UASQ site, thereby tuming on expression of the ⁇ -galactosidase genes operatively linked to that site.
  • untransformed yeast cells showed essentially no ⁇ -galactosidase adivity (essentially no color reaction), as did cells harboring plasmid pHP236.
  • the latter cells produced a hybrid regulatory protein which, due to the presence of the fundional HIV-1 protease between the DNA-binding and transcription-adivating domains, underwent autoproteolysis.
  • an intad transcription adivator was not available in these cells to stimulate ⁇ -galadosidase produdion at the UASQ site.
  • MUG units was obtained by subtrading fluorescence values obtained with the MUG reagent mixture only from all values measured. Had a spedfic inhibitor of the HIV-1 protease been present in the cells containing plasmid pHP236, autoproteolysis would have been prevented, the hybrid regulatory protein would have remained intad, and the result would have been markedly increased ⁇ -galacto- sidase adivity in both assays.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Protéines régulatrices hybrides contenant des séquences d'acides aminés susceptibles d'être clivées par des enzymes protéolitiques spécifiques. Lorsque lesdites enzymes agissent sur les protéines régulatrices hybrides, ces dernières sont rendues moins actives, modifiant ainsi la vitesse de production de produits de gènes indicateurs régulés par les protéines régulatrices. L'invention concerne également des ADN codant lesdites protéines régulatrices, des vecteurs recombinés et des cellules eukariotes transformées contenant lesdits ADN, ainsi que des procédés d'identifications d'inhibiteurs des enzymes protéolitiques spécifiques.
PCT/US1991/002283 1990-04-13 1991-04-10 Nouveaux dosages de proteases WO1991016436A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US50900790A 1990-04-13 1990-04-13
US509,007 1990-04-13

Publications (1)

Publication Number Publication Date
WO1991016436A1 true WO1991016436A1 (fr) 1991-10-31

Family

ID=24024942

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/002283 WO1991016436A1 (fr) 1990-04-13 1991-04-10 Nouveaux dosages de proteases

Country Status (2)

Country Link
AU (1) AU7742691A (fr)
WO (1) WO1991016436A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0631620A1 (fr) * 1992-03-16 1995-01-04 WOHLSTADTER, Jacob Nathaniel Procedes de selection
EP0781351A1 (fr) * 1994-09-16 1997-07-02 Sepracor, Inc. Procede in vitro pour predire la reponse evolutive de la protease du vih a un medicament le ciblant
WO1999038961A1 (fr) * 1998-01-30 1999-08-05 Sepracor Inc. Proteines de fusion regulatrices de genes et leurs procedes d'utilisation pour determiner la resistance d'une proteine a un medicament dirige contre elle
US5948620A (en) * 1996-08-05 1999-09-07 Amersham International Plc Reverse two-hybrid system employing post-translation signal modulation
WO2000012727A1 (fr) * 1998-08-31 2000-03-09 Vertex Pharmaceuticals Incorporated Proteines de fusion, molecules d'adn, vecteurs et cellules hotes utiles pour la mesure de l'activite protease
WO2001049731A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, proteine de regulation 9 de la protease humaine, et polynucleotide codant pour ce polypeptide
WO2001066585A1 (fr) * 2000-03-07 2001-09-13 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine 10 regulant la protease, et polynucleotide codant pour ce polypeptide
EP1159401A1 (fr) * 1999-03-05 2001-12-05 Glaxo Group Limited Essai sur les inhibiteurs du ftsh

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4833080A (en) * 1985-12-12 1989-05-23 President And Fellows Of Harvard College Regulation of eucaryotic gene expression
EP0321973A2 (fr) * 1987-12-23 1989-06-28 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Expression de la protéase P2A de HRV2 codée par un virus
WO1990006369A1 (fr) * 1988-12-09 1990-06-14 Smithkline Beecham Corporation Utilisation d'un site de clivage de protease de vih pour exprimer des proteines matures

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4833080A (en) * 1985-12-12 1989-05-23 President And Fellows Of Harvard College Regulation of eucaryotic gene expression
EP0321973A2 (fr) * 1987-12-23 1989-06-28 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Expression de la protéase P2A de HRV2 codée par un virus
WO1990006369A1 (fr) * 1988-12-09 1990-06-14 Smithkline Beecham Corporation Utilisation d'un site de clivage de protease de vih pour exprimer des proteines matures

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 335, 20 October 1988, London, GB., PTASHNE MARK, "How Eucaryotic Transcriptional Activators Work", pages 683-689. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0631620A1 (fr) * 1992-03-16 1995-01-04 WOHLSTADTER, Jacob Nathaniel Procedes de selection
EP0631620A4 (fr) * 1992-03-16 1997-11-12 Jacob Nathaniel Wohlstadter Procedes de selection.
EP0781351A1 (fr) * 1994-09-16 1997-07-02 Sepracor, Inc. Procede in vitro pour predire la reponse evolutive de la protease du vih a un medicament le ciblant
EP0781351A4 (fr) * 1994-09-16 2002-05-08 Sepracor Inc Procede in vitro pour predire la reponse evolutive de la protease du vih a un medicament le ciblant
US5948620A (en) * 1996-08-05 1999-09-07 Amersham International Plc Reverse two-hybrid system employing post-translation signal modulation
WO1999038961A1 (fr) * 1998-01-30 1999-08-05 Sepracor Inc. Proteines de fusion regulatrices de genes et leurs procedes d'utilisation pour determiner la resistance d'une proteine a un medicament dirige contre elle
US6117639A (en) * 1998-08-31 2000-09-12 Vertex Pharmaceuticals Incorporated Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity
WO2000012727A1 (fr) * 1998-08-31 2000-03-09 Vertex Pharmaceuticals Incorporated Proteines de fusion, molecules d'adn, vecteurs et cellules hotes utiles pour la mesure de l'activite protease
JP2002523102A (ja) * 1998-08-31 2002-07-30 バーテックス ファーマシューティカルズ インコーポレイテッド プロテアーゼ活性を測定するために有用な融合タンパク質、ｄｎａ分子、ベクター、および宿主細胞
US6528276B1 (en) 1998-08-31 2003-03-04 Vertex Pharmaceuticals Inc. Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity
EP1159401A1 (fr) * 1999-03-05 2001-12-05 Glaxo Group Limited Essai sur les inhibiteurs du ftsh
EP1159401A4 (fr) * 1999-03-05 2002-11-06 Glaxo Group Ltd Essai sur les inhibiteurs du ftsh
WO2001049731A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, proteine de regulation 9 de la protease humaine, et polynucleotide codant pour ce polypeptide
WO2001066585A1 (fr) * 2000-03-07 2001-09-13 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine 10 regulant la protease, et polynucleotide codant pour ce polypeptide

Also Published As

Publication number Publication date
AU7742691A (en) 1991-11-11

Similar Documents

Publication Publication Date Title
US5599906A (en) Protease assays
Zybarth et al. Domains upstream of the protease (PR) in human immunodeficiency virus type 1 Gag-Pol influence PR autoprocessing
US5916804A (en) Method for site-specific integration of nucleic acids and related products
AU636383B2 (en) Screening methods for protease inhibitors
Sadowski et al. GAL4 is phosphorylated as a consequence of transcriptional activation.
US10221422B2 (en) Blue light-inducible system for gene expression
US6063562A (en) In vitro method for predicting the evolutionary response of HIV protease to a drug targeted thereagainst
Dasmahapatra et al. A genetic system for studying the activity of a proteolytic enzyme.
US5322801A (en) Protein partner screening assays and uses thereof
BLAIR et al. Utilization of a mammalian cell-based RNA binding assay to characterize the RNA binding properties of picornavirus 3C proteinases
Baum et al. beta-Galactosidase containing a human immunodeficiency virus protease cleavage site is cleaved and inactivated by human immunodeficiency virus protease.
EP0646644B1 (fr) Système lié à la membrane périplasmic pour la détection des interactions protéine-protéine
WO1991016436A1 (fr) Nouveaux dosages de proteases
JPH11506608A (ja) 細胞傷害性に基づく遺伝的選択システム（ｔｏｘｓｅｌ）
JPS63309185A (ja) ウイルス特異的遺伝子コントロール下にインジケータ遺伝子産生物を発現する安定なヒト細胞系
US20030049712A1 (en) Method of detecting protease activity in a cell
US5948620A (en) Reverse two-hybrid system employing post-translation signal modulation
Xiang et al. Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation
US5776675A (en) Identification of compounds modulating protein/cell membrane association
JPH04500602A (ja) 合成ｈｉｖプロテアーゼ遺伝子およびその発現法
US5532124A (en) Genetically engineered bacteria to identify and produce medically important agents
AU2217800A (en) Methods and compositions for identifying protease modulators
WO1999011801A2 (fr) Systemes de commutation de genes a base de proteases
Murray et al. Inactivation of a yeast transactivator by the fused HIV-1 proteinase: a simple assay for inhibitors of the viral enzyme activity
EP0607311A1 (fr) Procedes et compositions d'identification d'inhibiteurs de la replication du virus du papillome

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR CA FI HU JP KP KR LK MC MG MW NO RO SD SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CM DE DK ES FR GA GB GR IT LU ML MR NL SE SN TD TG

NENP Non-entry into the national phase

Ref country code: CA

ENP Entry into the national phase

Ref country code: US

Ref document number: 1997 989789

Date of ref document: 19971212

Kind code of ref document: A

Format of ref document f/p: F