EP1015607A1 - Adn codant une proteine ras de traitement a terminaison carboxyle - Google Patents

Adn codant une proteine ras de traitement a terminaison carboxyle

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Publication number
EP1015607A1
EP1015607A1 EP98947194A EP98947194A EP1015607A1 EP 1015607 A1 EP1015607 A1 EP 1015607A1 EP 98947194 A EP98947194 A EP 98947194A EP 98947194 A EP98947194 A EP 98947194A EP 1015607 A1 EP1015607 A1 EP 1015607A1
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EP
European Patent Office
Prior art keywords
protein
ras
terminal processing
ras carboxyl
carboxyl
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP98947194A
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German (de)
English (en)
Inventor
Francis Farrell
Helena Chamberlain
Dana Johnson
Lehka Patel
Linda Jolliffe
Jose Galindo
Arne Huvar
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Janssen Pharmaceuticals Inc
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Ortho McNeil Pharmaceutical Inc
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Publication of EP1015607A1 publication Critical patent/EP1015607A1/fr
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • Ras protein is a 21kDa. guanosine triphosphate (GTP) binding protein that serves as a primary regulator of cell growth and differentiation. It cycles between an active, GTP bound form and an inactive GDP form. Ras is oncogenic when it is constitutively active due to a mutation that locks the protein in its GTP state (1). Ras has been found in 50 % of colorectal and 95% of pancreatic cancers (2). Ras becomes membrane associated, requisite to exert its biological effect, upon three posttranslational carboxyl terminal modifications
  • Ras proteins terminate in a CAAX motif where C is cysteine, A is an aliphatic amino acid, and X is any amino acid.
  • the first step is the addition of the lipid moiety farnesyl at the cysteine residue via a thioether bond. This is followed by the removal of the three COOH-terminal amino acids and the addition of a methyl group on the newly exposed carboxyl prenyl cysteine. These three modifications are imperative for transformation activity.
  • the yeast gene responsible for removal of the three terminal amino acids has been identified (4). Deletion of the gene was shown to interrupt ras function, namely ras localization and signaling. We describe herein the identification of a novel human ras protease isolated by a homology search to the yeast amino acid sequence. Inhibition of the human ras protease should be efficacious for cancer treatment.
  • a DNA molecule encoding a ras carboxyl-terminal processing protein has been cloned and characterized and it represents a novel class of ras processing enzymes.
  • DNA molecules encoding the ras carboxyl-terminal processing protein have been isolated.
  • the biological and structural properties of these proteins are disclosed, as is the amino acid and nucleotide sequence.
  • the recombinant protein is useful to identify modulators of the ras carboxyl-terminal processing protein. Modulators identified in the assay disclosed herein are useful as therapeutic agents.
  • the recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules.
  • Figure 1 The nucleotide sequence of human ras carboxyl terminal processing protein is shown.
  • Figure 2 The amino acid sequence of human ras carboxyl terminal processing protein is shown.
  • Figure 3 Northern blot analysis of human ras carboxyl terminal processing protein expression
  • Figure 4 The activity of the recombinantly produced human ras carboxyl terminal processing protein is shown.
  • Figure 5 The identification of the enzymatic product of the active recombinant human ras carboxyl terminal processing protein is shown by HPLC.
  • Figure 6 The map of the plasmid containing the recombinant human ras carboxyl terminal processing protein-encoding DNA is shown.
  • the present invention relates to DNA encoding ras carboxyl-terminal processing protein which was isolated from ras carboxyl-terminal processing protein producing cells.
  • Ras carboxyl-terminal processing protein refers to protein which can specifically function as a ras modulator.
  • Vertebrate cells capable of producing ras carboxyl-terminal processing protein include: heart, brain, liver, skeletal muscle, placenta, lung, kidney, and pancreas as shown by the ability of a ras carboxyl- terminal processing protein cDNA to hybridize to polyA+ RNA isolated from these cells.
  • ras carboxyl-terminal processing protein RNA is detected in human cancer cell lines including HL-60, HeLa, K562, Molt-4, Burkitts Lymphoma Raji, colorectal adenocarcinoma, SW480, lung carcinoma A549 and melanoma, G361 as described above.
  • ras carboxyl-terminal processing protein cDNA may also be suitable for use to isolate ras carboxyl- terminal processing protein cDNA. Selection of suitable cells may be done by screening for ras carboxyl-terminal processing protein activity in cell extracts or northern analysis to detect mRNA. Activity may be assessed by detecting processing activity on a ras carboxyl terminus substrate. Cells which possess ras carboxyl- terminal processing protein activity in this assay may be suitable for the isolation of ras carboxyl-terminal processing protein DNA or mRNA. Any of a variety of procedures known in the art may be used to molecularly clone ras carboxyl-terminal processing protein DNA.
  • ras carboxyl-terminal processing protein genes following the construction of a ras carboxyl-terminal processing protein -containing cDNA library in an appropriate expression vector system.
  • Another method is to screen ras carboxyl-terminal processing protein-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the ras carboxyl- terminal processing protein subunits.
  • An additional method consists of screening a ras carboxyl-terminal processing protein-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding ras carboxyl- terminal processing protein.
  • This partial cDNA is obtained by the specific PCR amplification of ras carboxyl-terminal processing protein DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence of the purified ras carboxyl-terminal processing protein.
  • Another method is to isolate RNA from ras carboxyl-terminal processing protein-producing cells and translate the RNA into protein via an in vitro or an in vivo translation system.
  • RNA into a peptide or protein will result in the production of at least a portion of the ras carboxyl-terminal processing protein which can be identified by, for example, immunological reactivity with an anti-ras carboxyl-terminal processing protein antibody or by biological activity of ras carboxyl-terminal processing protein.
  • pools of RNA isolated from ras carboxyl-terminal processing protein-producing cells can be analyzed for the presence of an RNA which encodes at least a portion of the ras carboxyl-terminal processing protein. Further fractionation of the RNA pool can be done to purify the ras carboxyl- terminal processing protein RNA from non- ras carboxyl-terminal processing protein RNA.
  • the peptide or protein produced by this method may be analyzed to provide amino acid sequences which in turn are used to provide primers for production of ras carboxyl-terminal processing protein cDNA, or the RNA used for translation can be analyzed to provide nucleotide sequences encoding ras carboxyl-terminal processing protein and produce probes for this production of ras carboxyl-terminal processing protein cDNA.
  • This method is known in the art and can be found in, for example, Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989.
  • libraries as well as libraries constructed from other cells or cell types, may be useful for isolating ras carboxyl-terminal processing protein-encoding DNA.
  • Other types of libraries include, but are not limited to, cDNA libraries derived from other cells, from organisms other than humans, and genomic DNA libraries that include YAC (yeast artificial chromosome) and cosmid libraries.
  • cDN A libraries may be prepared from cells or cell lines which have ras carboxyl-terminal processing protein activity.
  • the selection of cells or cell lines for use in preparing a cDNA library to isolate ras carboxyl-terminal processing protein cDNA may be done by first measuring cell associated ras carboxyl-terminal processing protein activity using a ras carboxyl terminal substrate.
  • cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring
  • DNA encoding ras carboxyl-terminal processing protein may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techiques can be found in Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). In order to clone the ras carboxyl-terminal processing protein gene by the above methods, the amino acid sequence of ras carboxyl-terminal processing protein may be necessary.
  • ras carboxyl-terminal processing protein may be purified and partial amino acid sequence determined by automated sequenators. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids from the protein is determined for the production of primers for PCR amplification of a partial ras carboxyl-terminal processing protein DNA fragment.
  • the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides.
  • Only one member of the set will be identical to the ras carboxyl-terminal processing protein sequence but will be capable of hybridizing to ras carboxyl-terminal processing protein DNA even in the presence of DNA oligonucleotides with mismatches.
  • the mismatched DNA oligonucleotides may still sufficiently hybridize to the ras carboxyl- terminal processing protein DNA to permit identification and isolation of ras carboxyl-terminal processing protein encoding DNA.
  • DNA isolated by these methods can be used to screen DNA libraries from a variety of cell types, from invertebrate and vertebrate sources, and to isolate homologous genes.
  • Purified biologically active ras carboxyl-terminal processing protein may have several different physical forms.
  • Ras carboxyl-terminal processing protein may exist as a full-length nascent or unprocessed polypeptide, or as partially processed polypeptides or combinations of processed polypeptides.
  • the full-length nascent ras carboxyl-terminal processing protein polypeptide may be postranslationally modified by specific proteolytic cleavage events which result in the formation of fragments of the full length nascent polypeptide.
  • a fragment, or physical association of fragments may have the full biological activity associated with ras carboxyl-terminal processing protein however, the degree of ras carboxyl-terminal processing protein activity may vary between individual ras carboxyl-terminal processing protein fragments and physically associated ras carboxyl-terminal processing protein polypeptide fragments.
  • the cloned ras carboxyl-terminal processing protein DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant ras carboxyl-terminal processing protein.
  • Techniques for such manipulations are fully described in Maniatis, T, et al., supra, and are well known in the art.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria including E. coli, bluegreen algae, plant cells, insect cells, fungal cells including yeast cells, and animal cells.
  • Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or bacteria- invertebrate cells.
  • An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses
  • a variety of mammalian expression vectors may be used to express recombinant ras carboxyl-terminal processing protein in mammalian cells.
  • mammalian expression vectors which may be suitable for recombinant ras carboxyl-terminal processing protein expression, include but are not limited to, pMAMneo (Clontech), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
  • a variety of bacterial expression vectors may be used to express recombinant ras carboxyl-terminal processing protein in bacterial cells.
  • Commercially available bacterial expression vectors which may be suitable for recombinant ras carboxyl- terminal processing protein expression include, but are not limited to pET vectors (Novagen), pQE vectors (Qiagen), pGEX (Pharmacia), and pTrcHis (Invitrogen).
  • a variety of fungal cell expression vectors may be used to express recombinant ras carboxyl-terminal processing protein in fungal cells such as yeast.
  • pYES2 Invitrogen
  • Pichia expression vector Invitrogen
  • a variety of insect cell expression vectors may be used to express recombinant ras carboxyl-terminal processing protein in insect cells.
  • Commercially available insect cell expression vectors which may be suitable for recombinant expression of ras carboxyl-terminal processing protein include but are not limited to pBlueBacII (Invitrogen), pVL1392 (Pharmingen), and pFastBacHT (GibcoBRL).
  • DNA encoding ras carboxyl-terminal processing protein may be cloned into an expression vector for expression in a recombinant host cell.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as R coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to drosophila and silkworm derived cell lines.
  • Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CN-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), ⁇ TH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-
  • the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, lipofection, and electroporation.
  • the expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce ras carboxyl-terminal processing protein. Identification of ras carboxyl- terminal processing protein expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-ras carboxyl-terminal processing protein antibodies, and the presence of host cell-associated ras carboxyl- terminal processing protein activity.
  • ras carboxyl-terminal processing protein DNA may also be performed using in vitro produced synthetic mRNA.
  • Synthetic mRNA or mRNA isolated from ras carboxyl-terminal processing protein producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being generally preferred.
  • ras carboxyl-terminal processing protein DNA molecules including, but not limited to, the following can be constructed: the full-length open reading frame of the ras carboxyl-terminal processing protein cDNA encoding the putative 338 amino acid protein from approximately base 6 to approximately base 1013 (these numbers correspond to first nucleotide of first methionine and last nucleotide before the first stop codon) and several constructs containing portions of the cDNA encoding ras carboxyl-terminal processing protein.
  • All constructs can be designed to contain none, all or portions of the 5' or the 3' untranslated region of ras carboxyl-terminal processing protein cDNA.
  • Ras carboxyl- terminal processing protein activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells.
  • this ras carboxyl-terminal processing protein DNA construct is transferred to a variety of expression vectors, for expression in host cells including, but not limited to, mammalian cells, baculovirus-infected insect cells, coli, and the yeast S. cerevisiae.
  • Host cell transfectants and microinjected oocytes may be used to assay both the levels of ras carboxyl-terminal processing protein activity and levels of ras carboxyl- terminal processing protein by the following methods.
  • this involves the co-transfection of one or possibly two or more plasmids, containing the ras carboxyl-terminal processing protein DNA encoding one or more fragments or subunits.
  • oocytes this involves the co-injection of synthetic RNAs for ras carboxyl-terminal processing protein.
  • cellular protein is metabolically labelled with, for example 35 S-methionine for 24 hours, after which cell lysates and cell culture supernatants are harvested and subjected to immunprecipitation with polyclonal antibodies directed against the ras carboxyl-terminal processing protein.
  • ras carboxyl-terminal processing protein activity involves the direct measurement of ras processing activity in whole cells transfected with ras carboxyl-terminal processing protein cDNA or oocytes injected with ras carboxyl-terminal processing protein mRNA.
  • Ras carboxyl-terminal processing protein activity is measured by specific ras processing of a synthetic ras carboxyl terminal substrate with extracts of the host cells expressing ras carboxyl-terminal processing protein DNA.
  • Levels of ras carboxyl-terminal processing protein in host cells are quantitated by immunoaffinity and/or ligand affinity techniques.
  • Cells expressing ras carboxyl- terminal processing protein can be assayed for the number of ras carboxyl-terminal processing protein molecules expressed by measuring expression levels with a specific antibodies to 35 S-methionine labelled or unlabelled ras carboxyl-terminal processing protein.
  • Labelled ras carboxyl-terminal processing protein is analyzed by SDS-PAGE.
  • Unlabelled ras carboxyl-terminal processing protein is detected by Western blotting, ELISA or RIA assays employing ras carboxyl-terminal processing protein specific antibodies.
  • the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the ras carboxyl-terminal processing protein sequence but will be capable of hybridizing to ras carboxyl-terminal processing protein DNA even in the presence of DNA oligonucleotides with mismatches under appropriate conditions. Under alternate conditions, the mismatched DNA oligonucleotides may still hybridize to the ras carboxyl-terminal processing protein DNA to permit identification and isolation of ras carboxyl-terminal processing protein encoding DNA.
  • DNA encoding ras carboxyl-terminal processing protein from a particular organism may be used to isolate and purify homologues of ras carboxyl-terminal processing protein from other organisms.
  • the first ras carboxyl- terminal processing protein DNA may be mixed with a sample containing DNA encoding homologues of ras carboxyl-terminal processing protein under appropriate hybridization conditions.
  • the hybridized DNA complex may be isolated and the DNA encoding the homologous DNA may be purified therefrom.
  • this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid.
  • a sequence bearing one or more replaced codons will be defined as a degenerate variation.
  • mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
  • DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally- occurring peptide.
  • Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
  • a “functional derivative” of ras carboxyl-terminal processing protein is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of ras carboxyl- terminal processing protein.
  • the term “functional derivatives” is intended to include the “fragments,” “variants,” “degenerate variants,” “analogs” and “homologues” or to "chemical derivatives” of ras carboxyl-terminal processing protein.
  • fragment is meant to refer to any polypeptide subset of ras carboxyl-terminal processing protein.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire ras carboxyl-terminal processing protein molecule or to a fragment thereof.
  • a molecule is "substantially similar” to ras carboxyl-terminal processing protein if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
  • analog refers to a molecule substantially similar in function to either the entire ras carboxyl-terminal processing protein molecule or to a fragment thereof.
  • ras carboxyl-terminal processing protein may be recovered to provide protein in active form.
  • ras carboxyl-terminal processing protein purification procedures are available and suitable for use including as described by Hitz and Georgopapadakou: [Hitz A. M. and Georgopapadakou N. H. (1996) FEBS Letters 391:310-312].
  • recombinant ras carboxyl-terminal processing protein may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
  • recombinant ras carboxyl-terminal processing protein can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full length nascent ras carboxyl- terminal processing protein, polypeptide fragments of ras carboxyl-terminal processing protein or ras carboxyl-terminal processing protein subunits.
  • ras carboxyl- terminal processing protein may be expressed as a fusion protein and purified by absorption to fusion moiety, e.g. GST fusion system [Smith, D. B. and Johnson, K. S.
  • Monospecific antibodies to ras carboxyl-terminal processing protein are purified from mammalian antisera containing antibodies reactive against ras carboxyl- terminal processing protein or are prepared as monoclonal antibodies reactive with ras carboxyl-terminal processing protein using the technique of Kohler and Milstein,
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for ras carboxyl-terminal processing protein.
  • Homogenous binding refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the ras carboxyl-terminal processing protein, as described above, ras carboxyl-terminal processing protein specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of ras carboxyl-terminal processing protein either with or without an immune adjuvant.
  • Preimmune serum is collected prior to the first immunization.
  • Each animal receives between about OJ mg and about 1000 mg of ras carboxyl-terminal processing protein associated with an acceptable immune adjuvant.
  • acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum- precipitate, water in oil emulsion containing Corvnebacterium parvum and tRNA.
  • the initial immunization consists of ras carboxyl-terminal processing protein in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both.
  • SC subcutaneously
  • IP intraperitoneally
  • Each animal is bled at regular intervals, preferably weekly, to determine antibody titer.
  • the animals may or may not receive booster injections following the initial immunizaiton. Those animals receiving booster injections are generally given an equal amount of the antigen in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20°C.
  • Monoclonal antibodies (mAb) reactive with ras carboxyl-terminal processing protein are prepared by immunizing inbred mice, preferably Balb/c, with ras carboxyl- terminal processing protein.
  • the mice are immunized by the IP or SC route with about OJ mg to about 10 mg, preferably about 1 mg, of ras carboxyl-terminal processing protein in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred.
  • the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks.
  • Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of ras carboxyl-terminal processing protein in a buffer solution such as phosphate buffered saline by the intravenous (IN) route.
  • Lymphocytes from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art.
  • Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas.
  • Fusion partners may include, but are not limited to: mouse myelomas P3/ ⁇ Sl/Ag 4-1; MPC-1 1; S-194 and Sp 2/0, with Sp 2/0 being generally preferred.
  • the antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%.
  • Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.
  • DMEM Dulbecco's Modified Eagles Medium
  • Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPLRA) using ras carboxyl-terminal processing protein as the antigen.
  • the culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
  • Monoclonal antibodies are produced in vivo by injection of pristane primed
  • Balb/c mice approximately 0.5 ml per mouse, with about 2 x 10° to about 6 x 10° hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
  • In vitro production of anti-ras carboxyl-terminal processing protein mAb is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art.
  • Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of ras carboxyl-terminal processing protein in body fluids or tissue and cell extracts.
  • monospecific antibodies may be utilized to produce antibodies specific for ras carboxyl-terminal processing protein polypeptide fragments, or full- length nascent ras carboxyl-terminal processing protein polypeptide, or the individual ras carboxyl-terminal processing protein subunits.
  • monospecific antibodies may be generated which are specific for only one ras carboxyl-terminal processing protein subunit or the fully functional protein.
  • Ras carboxyl-terminal processing protein antibody affinity columns are made by adding the antibodies to Affigel-10 (Biorad), a gel support which is activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support.
  • the antibodies are then coupled to the gel via amide bonds with the spacer arm.
  • the remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8).
  • the column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
  • the column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing ras carboxyl-terminal processing protein or ras carboxyl-terminal processing protein subunits are slowly passed through the column.
  • the column is then washed with phosphate buffered saline until the optical density (A2g ⁇ ) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6).
  • the purified ras carboxyl-terminal processing protein is then dialyzed against phosphate buffered saline.
  • DNA clones termed pHRP, are identified which encode proteins that, when expressed in bacteria, form a protein that can cleave the terminal three amino acids of ras protein.
  • the expression of ras carboxyl-terminal processing protein DNA results in the reconstitution of the properties observed in oocytes injected with ras carboxyl - terminal processing protein-encoding poly (A) + RNA.
  • Activated ras protein is associated with many malignant human tumors including breast, liver and colon . For ras to exert its function, it must be localized to the plasma membrane. The mechanism by which this localization occurs is by three distinct modifications to the immature protein.
  • the present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding ras carboxyl-terminal processing protein as well as the function of ras carboxyl-terminal processing protein in vivo.
  • Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
  • Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding ras carboxyl- terminal processing protein, or the function of ras carboxyl-terminal processing protein.
  • Compounds that modulate the expression of DNA or RNA encoding ras carboxyl-terminal processing protein or the function of ras carboxyl-terminal processing protein may be detected by a variety of assays.
  • the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
  • the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Modulators identified in this process are useful as therapeutic agents.
  • Kits containing ras carboxyl-terminal processing protein DNA or RNA, antibodies to ras carboxyl-terminal processing protein, or ras carboxyl-terminal processing protein may be prepared. Such kits are used to detect DNA which hybridizes to ras carboxyl-terminal processing protein DNA or to detect the presence of ras carboxyl-terminal processing protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses, diagnostic applications, and epidemiological studies.
  • the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of ras carboxyl- terminal processing protein DNA, ras carboxyl-terminal processing protein RNA or ras carboxyl-terminal processing protein.
  • the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of ras carboxyl-terminal processing protein activity or expression in tissue samples.
  • a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
  • the carrier would further comprise reagents such as recombinant ras carboxyl-terminal processing protein or anti-ras carboxyl-terminal processing protein antibodies suitable for detecting ras carboxyl-terminal processing protein.
  • the carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
  • Nucleotide sequences that are complementary to the ras carboxyl-terminal processing protein encoding DNA sequence can be synthesized for antisense therapy.
  • These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-
  • Ras carboxyl-terminal processing protein antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. Ras carboxyl-terminal processing protein antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce oncogenic ras activity.
  • compositions comprising ras carboxyl-terminal processing protein DNA, ras carboxyl-terminal processing protein RNA, or ras carboxyl-terminal processing protein, or modulators of ras carboxyl-terminal processing protein activity, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, or modulator.
  • compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders in which modulation of ras carboxyl-terminal processing activity is indicated.
  • the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • the pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
  • the term "chemical derivative” describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences. Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the ras carboxyl-terminal processing protein or its activity while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable.
  • the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
  • compositions containing compounds or modulators identified according to this invention as the active ingredient for use in the modulation of ras carboxyl-terminal processing protein can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
  • the compounds or modulators can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they may also be administered in intravenous
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per patient, per day.
  • the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0J, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day.
  • the dosages of the ras carboxyl- terminal processing protein modulators are adjusted when combined to achieve desired effects.
  • dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
  • compounds or modulators of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • compounds or modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • the active agents can be administered concurrently, or they each can be administered at separately staggered times.
  • the dosage regimen utilizing the compounds or modulators of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed.
  • a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
  • the compounds or modulators herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
  • carrier suitable pharmaceutical diluents, excipients or carriers
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
  • the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • Other dispersing agents which may be employed include glycerin and the like.
  • sterile suspensions and solutions are desired.
  • Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
  • Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
  • carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
  • the compounds or modulators of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds or modulators of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
  • the compounds or modulators of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • the compounds or modulators may be administered in capsule, tablet, or bolus form or alternatively they can be mixed in the animals feed.
  • the capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or
  • unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained.
  • An inert ingredient is one that will not react with the compounds or modulators and which is non-toxic to the animal being treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like.
  • These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection.
  • the active ingredient may also be administered as an additive to the feed by simply mixing the compound with the feedstuff or by applying the compound to the surface of the feed.
  • the active ingredient may be mixed with an inert carrier and the resulting composition may then either be mixed with the feed or fed directly to the animal.
  • Suitable inert carriers include corn meal, citrus meal, fermentation residues, soya grits, dried grains and the like.
  • the active ingredients are intimately mixed with these inert carriers by grinding, stirring, milling, or tumbling such that the final composition contains from 0.001 to 5% by weight of the active ingredient.
  • the compounds or modulators may alternatively be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraruminal, intratracheal, or subcutaneous.
  • the injectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier.
  • Acceptable liquid carriers include the vegetable oils such as peanut oil, cotton seed oil, sesame oil and the like as well as organic solvents such as solketal, glycerol formal and the like.
  • aqueous parenteral formulations may also be used.
  • the vegetable oils are the preferred liquid carriers.
  • the formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient.
  • Topical application of the compounds or modulators is possible through the use of a liquid drench or a shampoo containing the instant compounds or modulators as an aqueous solution or suspension.
  • These formulations generally contain a suspending agent such as bentonite and normally will also contain an antifoaming agent.
  • Formulations containing from 0.005 to 10% by weight of the active ingredient are acceptable.
  • Preferred formulations are those containing from 0.01 to 5% by weight of the instant compounds or modulators.
  • the Human Multiple Tissue Northern (MTNTM) Blot (catalog # 7760-1) contained approximately 2 ⁇ g of poly A+ RNA per lane from eight different human tissues, run on a denaturing formaldehyde 1.2% agarose gel, and transferred to a charge-modified nylon membrane. Lanes 1-8 contain, in order, RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas.
  • the Human Cancer Cell Line Multiple Tissue Northern (MTNTM) Blot contained approximately 2 ⁇ g of poly A+ RNA per lane from eight different human cell lines, run on a denaturing formaldehyde 1.2% agarose gel, and transferred to a charge-modified nylon membrane.
  • Lanes 1-8 contain, in order, RNA from human promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361.
  • Probes were labeled with ⁇ - 32 P-dCTP (Amersham, catalog # AA0005) as per protocol with Rediprime DNA labelling system (Amersham. catalog # RPN1633). 25ng of pHRP cDNA (928 bp PCR fragment corresponding to coding region of HRP) was labeled as above and hypridized as per protocol in the Clontech Multiple Tissue
  • 1786569 In aligning thel786569 contig sequence with yeast RCEl, a 49.8% nucleotide identity was revealed (Gap analysis in GCG). Based on nucleotide and amino acid alignments between the two, the 1786569 contig was not a full length sequence. It was missing approximately 500bp on the 5' end, based on the yeast RCEl sequence. The first 404 bp of the contig corresponds to the last 404 bp of the yeast RCEl gene. The rest of the contig (486 bp) is 3' untranslated sequence. Five reverse strand PCR primers were designed to the 1786569 contig.
  • a ⁇ gtlO human colorectal adenocarcinoma 5' stretch cDNA library was obtained from Clontech (catalog # HL3014a), and also ⁇ gtlO 5' and 3' amplimers. These amplimers along with the five reverse strand primers to thel 786569 contig were used to amplify several fragments within a 1400bp region (from the beginning of the putative HRP gene to the end of the 3' untranslated sequence). These fragments were gel isolated using the Geneclean II Kit (Bio 101, Inc.) and filled in (Large fragment of DNA polymerase, BRL) and kinased (T4 DNA Kinase, BRL). These products were then ligated as below. The coding region of the putative HRP clone had 41% identity in nucleotide sequence and 30% identity in amino acid sequence (Gap analysis in GCG).
  • RACE Rapid Amplification of cDNA ends
  • This first-round PCR product was used in a second-round PCR (same conditions) with a nested gene specific oligo. These PCR products were TA cloned (Invitrogen) and sequenced. Partially truncated clones were obtained because of high GC rich regions upstream of the ORF.
  • the Marathon kit (Clontech) was then used to perform another round of 5' race using prostate mRNA as a starting material. The kit was used according to the manufacturer's specifications.
  • the plasmid PCR-blunt was obtained from Invitrogen as part of the Zero-Blunt "PCR Cloning Kit (catalog # K2700-20). It was used initially as a cloning vector to insert the 1400 bp fragment from the purified PCR product from the nested PCR reactions. PCR products were ligated using 25ng PCR-blunt vector and approximately lOOng insert.
  • Ligation reaction proceeded at 12°C overnight with Gibco/ BRL T4 DNA ligase (1U) and 5X ligase buffer. Ligations were transformed into Top 10 cells as per protocol accompanying Zeroblunt kit. Colonies were confirmed as containing the correct insert by PCR, and grown in LB broth containing Kanamycin (50 ⁇ gl ⁇ ). DNA was isolated from the cultures with the use of a Qiagen tip- 100 Plasmid Maxi DNA isolation kit. DNA was sequenced by ACGT, Inc. (Northbrook, IL). HRP DNA coding region only was amplified again by PCR, with primers containing the restriction sites for Ndel on the 5' end and BamHI on the 3' end.DNA.
  • the PCR products were cloned again into PCR-blunt as above.
  • the PCR-blunt HRP constructs were digested with restriction enzymes Ndel and BamHI (BRL) to release the insert, which was gel isolated as above.
  • the resulting HRP fragments with digested ends were then ligated with pET 16b (Novagen) which had been digested with Ndel and BamHI.
  • the ligations were transformed into BL21 (DE3) cells (Novagen) as per protocol.
  • Xenopus laevis oocytes are prepared and injected using standard methods previously described and known in the art.
  • Adult female Xenopus laevis are anesthetized with 0.17% tricaine methanesulfonate and the ovaries are surgically removed and placed in a dish consisting of (mM): NaCl 82.5, KC1 2, MgCl 2 1, CaCl 2 1.8, HEPES 5 adjusted to pH 7.5 with NaOH (OR-2).
  • Ovarian lobes are broken open, rinsed several times, and gently shaken in OR-2 containing 0.2% collagenase (Sigma, Type 1A) for 2-5 hours.
  • Stage V and VI oocytes are selected and placed in media consisting of (mM): NaCl 86, KC1 2, MgG 2 1, CaQ 2 1 -8,
  • HEPES 5 Na pyruvate 2.5, theophylline 0.5, gentamicin 0J adjusted to pH 7.5 with NaOH (ND-96) for 24-48 hours before injection.
  • Oocytes are injected with 50-70 nl of poly(A + ) RNA (1 mg/ml) or 50 nl of ras carboxyl terminal processing protein RNA (0.1- 100 ng).
  • Control oocytes are injected with 50 nl of water. Oocytes are incubated for 2-10 days in ND-96 before analysis for expression of ras carboxyl terminal processing protein.
  • Incubations and collagenase digestion are carried out at 18°C.
  • Xenopus oocytes injected with ras carboxyl terminal processing protein poly(A) + RNA exhibit activity toward a synthetic ras carboxyl terminal substrate.
  • the nucleotide sequences of pHRP revealed single large open reading frame of about 1013 base pairs (FIGURE 1).
  • the cDNAs have 5' and 3 '-untranslated extensions of about 6 and about 331 nucleotides for pHRP.
  • the first in-frame methionine was designated as the initiation codon for an open reading frame that predicts a 338 amino acid protein with an estimated molecular mass (M r ) of about 40,000 Da (FIGURE 2).
  • the predicted ras carboxyl-terminal processing protein was aligned with nucleotide and protein databases and found to be related to a yeast ras converting enzyme (RCEl, GenBank accessionZ49260). Approximately 30% of the amino acids in ras carboxyl-terminal processing protein were identical RCEl.
  • Recombinant ras carboxyl terminal processing protein is produced in R coH following the transfer of the ras carboxyl terminal processing protein expression cassette into R coH expression vectors, including but not limited to, the pET series (Novagen).
  • the pET vectors place ras carboxyl terminal processing protein expression under control of the tightly regulated bacteriophage T7 promoter.
  • expression of ras carboxyl terminal processing protein is induced when an appropriate lac substrate (IPTG) is added to the culture.
  • the levels of expressed ras carboxyl terminal processing protein are determined by the assays described herein.
  • the coding sequence of HRP (nt 6-1019, encoding 338 amino acids) was cloned and expressed using the Ecdysone-Inducible Expression System (Invitrogen). Mammalian cell lines EcR-3T3 and EcR-293 (which are stably transfected with the regulator vector pVgRXR) were stably transfected under the selection of neomycin, with the coding sequence of HRP cloned into the Kpn I and EcoRI sites of the expression vector pIND (Spl)/V5-His A.
  • HNTG lysis buffer 50mM HEPES, pH 7.5, 150mM NaCl, 1% Triton X-100, 8% glycerol, 1.5mM MgCl 2 , 2mM EDTA. Cells lysates were spun for 10 minutes at 14,000 rpm and supernatants and pellets collected. Protein concentration was determined for each supernatant and 50 ⁇ g of protein from each supernatant sample and solubilized pellets were analyzed on 8%
  • Tris/glycine/SDS gels Tris/glycine/SDS gels. Western blots were performed and blots probed with an anti-6X His antibody (Invitrogen) and an antipeptide antibody generated against the C-terminus of the protein. Untransfected cells showed no expression of 6X His. Several transfected clonal isolates were positive for HRP expression by an antipeptide antibody. These cell lines were analyzed for HRP activity by two methods as described herein.
  • the ras carboxyl terminal processing protein cDNA can be cloned into the mammalian expression vector pcDNA3 as a Hindlll/BamHI fragment generated by PCR from pHRP. Recombinants were isolated, designated pcDNA3HRP and used to transfect mammalian cells (L-cells) by CaPO ⁇ DNA precipitation. Stable cell clones were selected by growth in the presence of G418. Single G418 resistant clones were isolated and shown to contain the intact ras carboxyl terminal processing protein gene.
  • Clones containing the ras carboxyl terminal processing protein cDNA are analyzed for expression using immunological techniques, such as immuneprecipitation, Western blot, and immunofluorescence using antibodies specific to the ras carboxyl terminal processing protein.
  • Antibody is obtained from rabbits innoculated with peptides that are synthesized from the amino acid sequence predicted from the ras carboxyl terminal processing protein sequences.
  • Cells that are expressing ras carboxyl terminal processing protein, stably or transiently are used to test for expression of ras carboxyl terminal processing protein and for ras processing activity. These cells are used to identify and examine other compounds for their ability to inhibit the ras carboxyl terminal processing protein.
  • Cassettes containing the ras carboxyl terminal processing protein cDNA in the positive orientation with respect to the promoter are ligated into appropriate restriction sites 3' of the promoter and identified by restriction site mapping and/or sequencing.
  • These cDNA expression vectors are introduced into fibroblastic host cells for example COS-7 (ATCC# CRL1651), and CV-1 tat [Sackevitz et al., Science 238: 1575 (1987)], 293, L (ATCC# CRL6362)] by standard methods including but not limited to electroporation, or chemical procedures (cationic liposomes, DEAE dextran, calcium phosphate).
  • Transfected cells and cell culture supernatants can be harvested and analyzed for ras carboxyl terminal processing protein expression as described herein.
  • All of the vectors used for mammalian transient expression can be used to establish stable cell lines expressing ras carboxyl terminal processing protein.
  • Unaltered ras carboxyl terminal processing protein cDNA constructs cloned into expression vectors are expected to program host cells to make ras carboxyl terminal processing protein.
  • Co-transfection of any vector containing ras carboxyl terminal processing protein cDNA with a drug selection plasmid including, but not limited to G418, aminoglycoside phosphotransferase; hygromycin, hygromycin-B phospholransferase; APRT, xanthine-guanine phosphoribosyl-transferase, will allow for the selection of stably transfected clones.
  • a drug selection plasmid including, but not limited to G418, aminoglycoside phosphotransferase; hygromycin, hygromycin-B phospholransferase; APRT, xanthine-guanine phosphoribosyl-transferase, will allow for the selection of stably transfected clones.
  • Levels of ras carboxyl terminal processing protein are quantitated by the assays described herein.
  • Ras carboxyl terminal processing protein cDNA constructs are also ligated into vectors containing amplifiable drug-resistance markers for the production of mammalian cell clones synthesizing the highest possible levels of ras carboxyl terminal processing protein. Following introduction of these constructs into cells, clones containing the plasmid are selected with the appropriate agent, and isolation of an over-expressing clone with a high copy number of plasmids is accomplished by selection in increasing doses of the agent.
  • ras carboxyl terminal processing protein The expression of recombinant ras carboxyl terminal processing protein is achieved by transfection of full-length ras carboxyl terminal processing protein cDNA into a mammalian host cell.
  • Baculovirus vectors which are derived from the genome of the AcNPV virus, are designed to provide high level expression of cDNA in the Sf9 line of insect cells
  • Recombinant baculoviruses expressing ras carboxyl terminal processing protein cDNA is produced by the following standard methods (InVitrogen Maxbac Manual): the ras carboxyl terminal processing protein cDNA constructs are ligated into the polyhedrin gene in a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P A., Nuc. Acid. Res. 18: 5667 (1990)] into Sf9 cells.
  • Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of ⁇ -galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555).
  • ras carboxyl terminal processing protein expression is measured by the assays described herein.
  • the cDNA encoding the entire open reading frame for ras carboxyl terminal processing protein is inserted into the BamHI site of pBlueBacII. Constructs in the positive orientation are identified by sequence analysis and used to transfect Sf9 cells in the presence of linear AcNPV mild type DNA.
  • Authentic, active ras carboxyl terminal processing protein is found in the cytoplasm of infected cells. Active ras carboxyl terminal processing protein is extracted from infected cells by hypotonic or detergent lysis.
  • Recombinant ras carboxyl terminal processing protein is produced in the yeast S. cerevisiae following the insertion of the cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins.
  • vectors such as EmBLyex4 or the like are ligated to the cistron [Rinas, U. et al., Biotechnology 8: 543-545 (1990); Horowitz B. et al., J. Biol.
  • the ras carboxyl terminal processing protein cistron is ligated into yeast expression vectors which fuse a secretion signal (a yeast or mammalian peptide) to the NH 2 terminus of the ras carboxyl terminal processing proteinfJacobson, M. A., Gene 85: 511-516 (1989); Riett L. and Bellon N. Biochem. 28: 2941-2949 (1989)].
  • a secretion signal a yeast or mammalian peptide
  • ras carboxyl terminal processing protein is expressed in yeast as a fusion protein conjugated to ubiquitin utilizing the vector pVEP [Ecker, D. J., J. Biol. Chem. 264: 7715-7719 (1989), Sabin, E. A., Biotechnology 7: 705-709 (1989), McDonnell D. P., Mol. Cell Biol. 9: 5517-5523 (1989)]
  • the levels of expressed ras carboxyl terminal processing protein are determined by the assays described herein.
  • Recombinantly produced ras carboxyl terminal processing protein may be purified by antibody affinity chromatography.
  • Ras carboxyl terminal processing protein antibody affinity columns are made by adding the anti- ras carboxyl terminal processing protein antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support.
  • the antibodies are then coupled to the gel via amide bonds with the spacer arm.
  • the remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8).
  • the column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
  • the column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents and the cell culture supernatants or cell extracts containing solubilized ras carboxyl terminal processing protein are slowly passed through the column.
  • the column is then washed with phosphate- buffered saline together with detergents until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6) together with detergents.
  • the purified ras carboxyl terminal processing protein is then dialyzed against phosphate buffered saline.
  • Ras carboxyl terminal processing protein is expressed as fusion protein with but not limited to purification tags including glutathione S-transferase (GST) and polyhistidine residues (His6) in tandem.
  • GST glutathione S-transferase
  • His6 polyhistidine residues
  • Cells or bacteria expressing protein are harvested and lysed by chemically and/or mechanical means. Lysates are applied to affinity matrix, glutathione sepharose and Ni 2+ charged agarose for GST and His6 fusion proteins respectively in buffered slurry. The affinity matrix is extensively washed and proteins are eluted by glutathione or imidazole for GST and His6 fusion proteins respectively.
  • Expression is monitered by wetern blot analysis using an antibody to fusion tag or an antibody to ras carboxyl terminal processing protein.
  • Figure 6 is a drawing of the plasmid that was used to produce the human ras carboxyl terminal protease-His6 fusion protein.
  • Protease activity is measured in microsomal preparations generated from transfected cell lines.
  • the method of preparation is adapted from Methods in Enzymology volume 96,88- 89. Briefly, cells are collected and extensively homogenized in a dounce homogenizer (type B pestle). The homogenate is centrifuged for 10 minutes at 1000 gav. The supernatant is collected and layered on a sucrose cushion and subjected to centrifugation for 30 minutes at 140,000 ga V in a Beckman TL ultracentrifuge (TLA 100.3 rotor). The resulting cell pellet is resuspended in a buffer containing 250 mM sucrose, 50 mM triethanolamine pH 7.5 and 1 mM DTT. Protein determination is performed and microsomes are stored at -70° C.
  • FRET Fluorescence Resonance Energy Transfer
  • a peptide substrate [peptide sequence: (Aedens) ESKTK (farnesyl) CVIM (Dabcyl) K- amide] was used corresponding to the C-terminus of K-rasB with fluorophore and chromophore groups attached to either end. 25 ⁇ M peptide was incubated with microsomal fraction (10 ⁇ g) generated from NLH3T3 HRP transfected cells (clone 5) and untransfected NTH3T3 cells in a buffer containing 200 mM Hepes pH 7.4, 100 mM NaCl and 5 mM MgCl .
  • a peptide substrate [peptide sequence: WKKKKSKTK (farnesyl)CVIM] was used corresponding to the C-terminus of K-ras with an N-terminal tip. 500 ⁇ M peptide was incubated with microsomes (4-20 ⁇ g total protein) in assay buffer (200mM Hepes, pH 1.4, lOOmM NaCl, 5mM MgCl 2 ) for 24 hours at 30° C. Proteins were precipitated and the isolated peptide run on HPLC to determine extent of cleavage.
  • the separation was performed on a Waters 625 HPLC fitted with a Water 996 Photodiode Array Detector using a Vydac Protein-Peptide C18 column (part number 218TP54) running a gradient of 0J % TFA in H20 (mobile phase A) to 0.07%TFA in acetonitrile (mobile phase B) consisting of 100 A for 10 min followed by a linear gradient of 0-100% B in 50 min, 5 min at 100%B, 100-0% B in 5 min and 5 min of 100% A. Proteins were precipitated by adding a two fold volume of 2% Zinc Sulfate in 50% acetonitrile prepared fresh daily from a stock of 4% Zinc sulfate and neat acetonitrile.

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Abstract

On a cloné et caractérisé de l'ADN codant la protéine RAS humaine de traitement à terminaison carboxyle. La protéine de recombinaison est capable de former une protéine biologiquement active. De l'ADNc a été exprimé dans des cellules hôtes de recombinaison qui produisent des protéines de recombinaison actives. La protéine de recombinaison est également purifiée à partir des cellules hôtes de recombinaison. En outre les cellules hôtes de recombinaison sont utilisées pour définir un procédé permettant d'identifier des modulateurs de l'activité enzymatique et des modulateurs sont identifiés.
EP98947194A 1997-09-19 1998-09-18 Adn codant une proteine ras de traitement a terminaison carboxyle Withdrawn EP1015607A1 (fr)

Applications Claiming Priority (3)

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US5940197P 1997-09-19 1997-09-19
US59401P 1997-09-19
PCT/US1998/019746 WO1999014343A1 (fr) 1997-09-19 1998-09-18 Adn codant une proteine ras de traitement a terminaison carboxyle

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EP1078076A2 (fr) * 1998-05-22 2001-02-28 Onyx Pharmaceuticals, Inc. Nouveaux acides nucleiques et polypeptides lies a une endopeptidase dirigee sur farnesyle
US6261793B1 (en) * 1999-03-04 2001-07-17 Schering Corporation RAS converting endoprotease (RCE) and methods
CN104718282A (zh) 2012-08-10 2015-06-17 Opx生物工艺学公司 用于生产脂肪酸和脂肪酸衍生产物的微生物及方法
US10047383B2 (en) 2013-03-15 2018-08-14 Cargill, Incorporated Bioproduction of chemicals
US11408013B2 (en) 2013-07-19 2022-08-09 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products
EP3022310B1 (fr) 2013-07-19 2019-10-16 Cargill, Incorporated Micro-organismes et procédés pour la production d'acides gras et de produits dérivés d'acides gras
EP2993228B1 (fr) 2014-09-02 2019-10-09 Cargill, Incorporated Production d'esters d'acides gras
CN110494566A (zh) 2017-02-02 2019-11-22 嘉吉公司 产生c6-c10脂肪酸衍生物的经遗传修饰的细胞

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JP2001516586A (ja) 2001-10-02
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WO1999014343A1 (fr) 1999-03-25

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