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WO1993021330A1 - Glutamine:fluctose-6-phosphate amidotransferase humaine - Google Patents

Glutamine:fluctose-6-phosphate amidotransferase humaine Download PDF

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Publication number
WO1993021330A1
WO1993021330A1 PCT/US1993/003773 US9303773W WO9321330A1 WO 1993021330 A1 WO1993021330 A1 WO 1993021330A1 US 9303773 W US9303773 W US 9303773W WO 9321330 A1 WO9321330 A1 WO 9321330A1
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WO
WIPO (PCT)
Prior art keywords
fructose
sequence
phosphate
glutamine
gfat
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PCT/US1993/003773
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English (en)
Inventor
Gary L. Mcknight
Paul O. Sheppard
Patrick J. O'hara
Patricia A. Mckernan
Robert A. Smith
Jeffrey Van Ness
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Zymogenetics, Inc.
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Publication of WO1993021330A1 publication Critical patent/WO1993021330A1/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
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • glucoregulatory effects of insulin are predominantly exerted in the liver, skeletal muscle and adipose tissue.
  • Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that result in, for example, the inhibition of glucose production and the stimulation of glucose utilization.
  • insulin stimulates glucose uptake and inhibits gluconeogenesis and glycogenolysis.
  • skeletal muscle and adipose tissue insulin acts to stimulate the uptake, storage and utilization of glucose.
  • the cellular mechanisms that result in the overall regulation of blood glucose levels are impaired, resulting in abnormally high levels of circulating insulin and glucose.
  • Type II non- insulin dependent diabetes also known as NIDDM and referred to hereinafter as Type II diabetes
  • Type II diabetes is characterized by abnormal basal and stimulated insulin secretion, increased endogenous hepatic glucose release and inefficient peripheral tissue glucose utilization, resulting in hyperinsulinemia and hyperglycemia.
  • Insulin resistance has also been documented in obesity, pregnancy, acromegaly, hypertension, atherosclerosis and certain catabolic situations associated with glucose intolerance. While insulin receptor mutations and decreased numbers of insulin receptors have been reported in patients exhibiting insulin resistance, such abnormalities in themselves do not explain the overall inability of insulin to stimulate glucose uptake in peripheral tissues such as in skeletal muscle and adipose tissue.
  • treatment regimes include body weight reduction, insulin administration and oral sulfonylureas.
  • Caloric restriction to achieve body weight reduction decreases the overall caloric intake and, more specifically, decreases glucose intake and reduces hepatic glycogen stores. Maintenance of a lower body weight results in a lower plasma glucose level.
  • Patients that derive the most benefit from body weight reduction are those with poor islet function and marked hyperglycemia.
  • Exogenous insulin administration serves to substitute for defective insulin secretion from islet cells.
  • Exogenous insulin serves to correct a hypoinsulinemic condition and relies on the ability of insulin to suppress hepatic glucose release and enhance peripheral glucose uptake.
  • Sulfonylurea administration serves to enhance insulin secretion and functions in the same way as exogenous insulin administration to lower plasma glucose levels.
  • the present invention fulfills this need by' providing materials and methods for use in detecting compounds capable of inhibiting GFAT activity and for preparing oligonucleotide probes capable of detecting glutamine:fructose-6-phosphate amidotransferase sequences.
  • the invention provides for the identification of compounds that inhibit GFAT activity through the use of an assay system employing recombinant human GFAT. Such compounds are useful, for example, in inhibiting endogenous GFAT activity and thereby inhibiting insulin resistance.
  • the present invention discloses isolated human glutamine:fructose-6-phosphate amidotransferase (GFAT) and isolated DNA molecules encoding human GFAT.
  • a representative human GFAT comprises the amino acid sequence of Sequence ID NO: 2 from methioinine, amino acid number 1, to glutamic acid, amino acid number 681.
  • representative DNA molecules encoding GFAT include the DNA sequence which comprises the nucleotide sequence shown in Sequence ID NO: 1 from nucleotide 123 to nucleotide 2165.
  • representative DNA molecules encoding GFAT encode the amino acid sequence of Sequence ID NO: 2 from methionine, amino acid number 1, to. glutamic acid, amino acid number 681.
  • an antiseru is obtained from an animal immunized with the human GFAT wherein the antiserum binds to human GFAT.
  • a monoclonal antibody against human GFAT is obtained.
  • DNA molecules of at least about 14 nucleotides are disclosed wherein the molecules are capable of hybridizing with a gene which encodes a human GFAT polypeptide and wherein the DNA molecule is at least 85% homologous to a corresponding DNA sequence of the human GFAT shown in Sequence ID NO: 1 or its complement.
  • the DNA molecules of at least about 14 nucleotides are labeled to provide a detectable signal.
  • DNA constructs containing the information necessary to direct the expression of GFAT are disclosed.
  • host cells containing DNA constructs containing information necessary for the expression of GFAT are disclosed.
  • methods for producing recombinant human GFAT are disclosed.
  • recombinant human GFAT is produced from cultured mammalian, bacterial or fungal cells according to the disclosed methods.
  • methods for detecting a compound which inhibits human GFAT are disclosed.
  • a test substance is exposed to human GFAT in the presence of fructose-6- phosphate and glutamine under physiological conditions and for a time sufficient to allow the test substance to inhibit GFAT activity, wherein a reduction in activity of the GFAT in comparison to the activity in the absence of the test substance is indicative of the presence in the test substance of a compound which inhibits human GFAT.
  • compounds which inhibit human GFAT are detected by measuring the production of radiolabeled glutamate in the presence of a test substance relative to the production of radiolabeled glutamate in the absence of the test substance.
  • a test substance is exposed to human GFAT in the presence of 3-acetylpyridine adenine dinucleotide, glutamate dehydrogenase, fructose- 6-phosphate and glutamine, and the 3-acetylpyridine adenine dinucleotide production is measured relative to the production of 3-acetylpyridine adenine dinucleotide in the absence of the test substance.
  • DNA construct A DNA molecule, or a clone of such a molecule, which has been constructed through human intervention to contain sequences arranged in a way that would not otherwise occur in nature.
  • Expression vectors are DNA constructs which contain, inter alia, a DNA sequence encoding a protein of interest together with a promoter and other sequences, such as a transcription terminator and polyadenylation signal, that facilitate expression of the protein. Expression vectors further contain genetic information that provides for their replication in a host cell, either by autonomous replication or by integration into the host genome. Examples of expression vectors commonly used for recombinant DNA are plasmids and certain viruses, although they may contain elements of both. They also may include one or more selectable markers. Transfection or transformation: The process of stably and hereditably altering the genotype of a recipient cell or microorganism by the introduction of isolated DNA. This is typically detected by a change in the phenotype of the recipient organism. The term “transformation” is generally applied to microorganisms, while “transfection” is generally used to describe this process in cells derived from multicellular organisms.
  • An object of the present invention is to provide methods for detecting GFAT inhibitors.
  • a feature of the present invention is an isolated DNA molecule encoding human GFAT. Such molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are naturally associated and may include naturally occurring 5• and 3 ' untranslated sequences that represent regulatory regions such as promoters and terminators. The identification of regulatory regions within the naturally occuring 5' and 3 1 untranslated regions will be evident to one of ordinary skill in the art (for review, see Dynan and Tijan, Nature 316: 774-778,.
  • the isolated DNA molecules of the present invention are useful in producing recombinant human GFAT.
  • the present invention provides the advantage that human GFAT is produced in high quantities that may be readily purified for use in the disclosed methods for detecting compounds capable of inhibiting GFAT activity.
  • GFAT the first enzyme in the hexosamine pathway, catalyzes the formation of glucosamine-6- phosphate from fructose-6-phosphate and glutamine.
  • DNA molecules encoding GFAT may be isolated using standard cloning methods such as those described by Maniatis et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, (1982) ; which is incorporated herein by reference) , Sambrook et al. (Molecular Cloning: 1
  • GFAT coding sequences may be synthesized using standard techniques that are well known in the art, such as by synthesis on a DNA synthesizer.
  • Sequence ID NO: 1 and Sequence ID NO: 2 disclose a representative nucleotide sequence and deduced amino acid sequence of human GFAT. Analysis of the sequence discloses a primary translation product of 681 amino acids. As will be recognized by those skilled in the art, minor variations in the amino acid sequence of GFAT may occur. Such variations may be due to, for example, genetic polymorphisms or minor proteolysis. Sequence variations may also be introduced by genetic engineering techniques.
  • DNA molecules encoding GFAT or portions thereof may be used, for example, to directly detect GFAT sequences in cells.
  • Such DNA molecules are generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise from about 14 nucleotides to about 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion or even the entire cDNA of a GFAT gene.
  • the synthetic oligonucleotides of the present invention are at least 85% homologous to a corresponding DNA sequence of the human glutamine:fructose-6-phosphate amidotransferase of Sequence ID NO: 1 or a complementary sequence thereto.
  • the molecules are labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle, etc. according to methods known in the art.
  • DNA molecules used within the present invention may be labeled and used in a hybridization procedure similar to the Southern or dot blot.
  • conditions that allow the DNA molecules of the present invention to hybridize to GFAT sequences or GFAT-like sequences may be determined by methods well known in the art and reviewed, for example, by Sambrook et al. (Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor, NY, 1989; which is incorporated herein by reference) .
  • hybridization conditions i.e. stringency of hybridization
  • human GFAT sequence variants may be identified using DNA molecules of the present invention and, for example, the polymerase chain reaction (PCR) (disclosed by Saiki et al., Science 239: 487, 1987; Mullis et al., U.S. Patent 4,686,195; Mullis et al., U.S. Patent 4,683,202, Orita et al., Proc. Nat'l Acad. Sci. USA 86: 2766-2770, 1989, Spinardi et al., Nucleic Acids Res.
  • PCR polymerase chain reaction
  • GFAT coding sequences are inserted into suitable expression vectors which are in turn introduced into prokaryotic and/or eukaryotic host cells.
  • Expression vectors for use in carrying out the present invention comprise a promoter capable of directing the transcription of a cloned DNA and a transcriptional terminator. In some circumstances, it may be preferable to direct GFAT into the secretory pathway of the host cell. In such a case, the expression vector would further comprise a secretory signal sequence capable of directing the secretion of the protein encoded by the cloned DNA downstream of the promoter and operably linked to the GFAT coding sequence.
  • the present invention also encompasses antisense oligonucleotides and "antisense" expression vectors capable of directing the transcription of antisense mRNA, which is complementary to mRNA encoding GFAT protein, capable of hybridizing to part or all of the endogenous GFAT-encoding mRNA.
  • antisense expression vectors thus transcribe sequences that are capable of preventing the translation of GFAT mRNA in a host cell thus reducing GFAT expression levels. It may be advantageous to utilize antisense sequences as described herein in pancreatic 5-cells to reduce GFAT expression levels.
  • Preferable sequences for use in antisense vectors are those sequences which inhibit the translation of GFAT mRNA in host cells that have been transfected or transformed with the antisense vector and include the 5* non-coding region of GFAT and the sequences that hybridize to the translation start AUG.
  • the antisense mRNA which corresponds to a GFAT DNA sequence, may contain less than the entire length of GFAT sequence and may contain nucleic acid changes that do not inhibit hybridization to GFAT mRNA but significantly reduce the translation of the mRNA into GFAT.
  • Antisense GFAT oligonucleotide sequences are preferably obtained from the 5 1 non-coding region and are preferably between 10 and 25 nucleotides in length, most preferably 18 nucleotides in length.
  • Antisense GFAT expression vectors may be prepared by inserting a GFAT sequence in the opposite orientation relative to the transcriptional promoter in the expression vectors discussed in detail herein. The selection of suitable promoters, terminators, and vector sequences are within the level of ordinary skill in the art (for review see Mirabelli et al., Anti-Cancer Drug Des. 6.: 647-661, ⁇ 1991; Crooke, Anti-Cancer Drug Des. 6_: 609-646, 1991; James, Antiviral Che . Chemothe .
  • Host cells for use in practicing the present invention include prokaryotic and eukaryotic cells.
  • Preferred prokaryotic host cells for use in carrying out the present invention are strains of . the bacteria E. coli, although Bacillus and other genera are also useful.
  • Eukaryotic host cells for use in the present invention include mammalian, avian, plant, insect, and fungal cells. Fungal cells, including species of yeast (e.g., Saccharo yces spp. , Schizosaccharomyces spp. ) or filamentous fungi (e.g., Aspergillus spp:, Neurospora spp.) may be used as host cells within the present invention. Strains of the yeast Saccharomyces cerevisiae are particularly preferred.
  • plasmids suitable for transforming bacteria include pBR322 (Bolivar et al. , Gene 2.: 95-113, 1977), the pUC plasmids (Messing, Meth. Enzvmol. 101:20-77, 1983), Vieira and Messing, Gene 19: 259-268, 1982), pCQV2 (Queen, J. Mol. APPI. Genet. 2 : 1- 10, 1983), pIC vectors (Marsh et al., Gene 32: 481-485, 1984) , and derivatives thereof.
  • Suitable vectors may be purchased from commercial suppliers (i.e., from GIBCO-BRL (Gaithersburg, MD) , Boehringer Mannheim (Indianapolis, IN), and New England Biolabs (Beverly, MA)).
  • Appropriate promoters include the trp (Nichols and Yanofsky, Meth. Enzvmol. 101: 155-164, 1983), lac (Casabadan et al., J. Bacteriol. 143: 971-980, 1980), and phage ⁇ promoter systems (Queen, ibid.).
  • a particularly preferred promoter is the tac promoter (Amann et al., Gene 40: 183, 1985 and de Boer et al., Proc. Natl. Acad. Sci. USA 80: 12, 1983).
  • Bacterial expression vectors will also include a ribosome-binding site upstream of the initiation codon. Ribosome-binding sites, also known as Shine-Delgarno sequences (Shine and Delgarno, Nature: -
  • a suitable bacterial host cell is well within the level of ordinary skill in the art. Techniques for transforming bacterial host cells and expressing foreign genes cloned in them are well known in the art (see e.g., Maniatis et al. , eds. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, 1982; and Sambrook et al., ibid.). Methods for the recovery of the proteins in biologically active forms from bacteria. are discussed in U.S. Patents Numbers 4,966,963 and 4,999,422, which are incorporated herein by reference.
  • yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEpl3 (Broach et al. , Gene 8: 121-133, 1979), POT vectors (Kawasaki et al, U.S. Patent No. 4,931,373, which is incorporated by reference herein) , pJDB249 and pJDB219 (Begg ⁇ , Nature275:104-108. 1978) and derivatives thereof.
  • Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibits a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.
  • selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include LEU2 (Broach et al., ibid.), URA3 (Botstein et al., Gene 8 . : 17, 1970), HIS3 (Struhl et al. , ibid.) or POT1 (Kawasaki et al. , ibid.).
  • Another suitable selectable marker is the chloramphenicol acetyl transferase (CAT) gene, which confers chloramphenicol resistance on yeast cells.
  • CAT chloramphenicol acetyl transferase
  • Preferred promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al. , ii
  • the expression units may also include a transcriptional terminator.
  • a preferred transcriptional terminator is the TPI1 terminator (Alber and Kawasaki, ibid.).
  • proteins of the present invention can be expressed in filamentous fungi, for example, strains of the fungi Aspergillus (McKnight et al., U.S. Patent No. 4,935,349, which is incorporated herein by reference) .
  • useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985) and the tpiA promoter.
  • An example of a suitable terminator is the ADH3 terminator (McKnight et al., ibid., 1985).
  • the expression units utilizing such components are cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus.
  • the genotype of the host cell will generally contain a genetic defect that is complemented by the selectable marker present on the expression vector. Choice of a particular host and ⁇ 1 selectable marker is well within the level of ordinary skill in the art.
  • E. coli To optimize production of recombinant human GFAT in host cells and to facilitate purification of the protein, it may be preferable to use host cells that are deficient in the native host GFAT analog.
  • E. coli for example, it may be preferable to use a host cell containing a genetic defect in the glmS gene, the glutamine synthetase gene.
  • E. coli strains carrying glmS ⁇ mutations have been described by Wu and Wu (J. Bact. 105: 455-466, 1971) and Dutka-Malen et al. (Biochimie 70: 287-290, 1988) among others.
  • Yeast strains defective in glutamine:fructose-6-phosphate amidotransferase activity may be obtained, for example, from the Yeast Genetic Stock Center (Department of Molecular and Cellular Biology, Division of Genetics, University of California at Berkeley) . It may be preferable to disrupt the E . . coli glmS gene or the Saccharomyces cerevisiae gcnl gene using methods well established in the literature (see Rothstein, Methods in Enzvmology 101: 202-211, 1981; which is incorporated herein by reference) . The DNA sequence encoding the glnS gene has been disclosed by, for example. Walker et al. (Biochem. J.
  • the host strain carries a mutation, such as the yeast pep4 mutation (Jones, Genetics 85: 23-33, 1977), which results in reduced proteolytic activity.
  • the host strain carries a mutation in the SSC1 gene (Smith et al., U.S. Patent 5,057,416; which is now referred to as PMR1; Rudolph et al., Cell 58: 133- 146, 1989) which results in the increase in secretion of heterologous proteins. It may be advantageous to disrupt the PMR1 gene.
  • yeast host cells For secretion of foreign genes from yeast host cells, it may also be preferable to utilize a host cell that contains a genetic deficiency in at least one gene required for asparagine-linked glycosylation of glycoproteins is used.
  • a genetic deficiency will be in either the MNN9 gene or the MNN1 gene or both (described in pending, commonly assigned U.S. Patent Application Serial No. 07/189,547, which is incorporated by reference herein in its entirety) .
  • the yeast host cell contains a disruption of both the MNN1 and MNN9 genes.
  • a yeast strain containing disruptions of both the MNN1 and MNN9 strains was deposited with the American Type Culture Collection (Rockville, MD) under Accession number 20996.
  • Yeast host cells having such defects may be prepared using standard techniques of mutation and selection. Ballou et al. (J. Biol. Chem. 255: 5986-5991, 1980) have described the isolation of annoprotein biosynthesis mutants that are defective in genes which affect asparagine-linked glycosylation. Briefly, mutagenized yeast cells were screened using fluoresceinated antibodies directed against the outer mannose chains present on wild-type yeast. Mutant cells that did not bind antibody were further characterized and were found to be defective in the addition of asparagine-linked oligosaccharide moieties. ⁇ -
  • cultured mammalian cells may be used as host cells within the present invention.
  • Preferred cultured mammalian cells for use in the present invention include the 3T3-L1 (ATCC CCL 92.1), COS-1 (ATCC CRL 1650), BHK, and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol.6: 59-72, 1977) cell lines.
  • a preferred BHK cell line is the BHK 570 cell line (deposited with the American Type Culture Collection under accession number CRL 10314) .
  • Rat Hep I ATCC CRL 1600
  • Rat Hep II ATCC CRL 1548
  • TCMK ATCC CCL 139
  • Human lung ATCC CCL 75.1
  • Human hepatoma ATCC HTB-52
  • Hep G2 ATCC HB 8065
  • Mouse liver ATCC CCL 2911
  • NCTC 1469 ATCC CCL 9.1
  • DUKX cells Urlaub and Chasin. Proc. Natl. Acad. Sci USA77: 4216-4220, 1980.
  • Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA.
  • Preferred promoters include viral promoters and cellular promoters.
  • Viral promoters include the immediate early cytomegalovirus promoter (Boshart et al. , Cell 41: 521-530, 1985) and the SV40 promoter (Subramani et al. , Mol. Cell. Biol. 1: 854-864, 1981) .
  • Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al. , U.S. Patent No.
  • telomere splice sites located downstream from the promoter and upstream from the DNA sequence encoding the peptide or protein of interest.
  • RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes.
  • a polyadenylation signal located downstream of the coding sequence of interest. Polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the Adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9.: 3719-3730, 1981) .
  • the expression vectors may include a noncoding viral leader sequence, such as the Adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites.
  • Preferred vectors may' also include enhancer sequences, such as the SV40 enhancer and the mouse ⁇ enhancer (Gillies, Cell 33: 717- 728, 1983) .
  • Expression vectors may also include sequences encoding the adenovirus VA RNAs.
  • Cloned DNA sequences may be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973; which are incorporated by reference herein in their entirety) .
  • Other techniques for introducing cloned DNA sequences into mammalian cells such as electroporation
  • a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.
  • Preferred selectable markers for use in cultured mammalian cells include genes that confer Iff resistance to drugs, such as neomycin, hygro ycin, and methotrexate.
  • the selectable marker may be an amplifiable selectable marker.
  • a preferred amplifiable selectable marker is the DHFR gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA, which is incorporated herein by reference) - The choice of selectable markers is well within the level of ordinary skill in the art.
  • Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Patent No. 4,713,339). It may also be advantageous to add additional DNA, known as "carrier DNA" to the mixture which is introduced into the cells.
  • Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.
  • Promoters, terminators and methods suitable for introducing expression vectors encoding GFAT into plant, avian and insect cells are well known in the art.
  • the use of baculoviruses for example, as vectors for expressing heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28: 215-224, 1990) .
  • the use of Agrobacterium rhizogenes as vectors for n expressing genes in plant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangalore) 11: 47-58, (1987)).
  • At least one signal sequence is operably linked to the DNA sequence of interest.
  • the choice of suitable signal sequences for a particular host cell is within the level of ordinary skill in the art.
  • Preferred signals for use in E. coli include the E. coli phoA signal sequence (Oka et al., Proc. Natl. Acad. Sci. USA 82: 7212, 1985).
  • preferred signal sequences include the alpha factor signal sequence (pre-pro sequence; Kurjan and Herskowitz, Cell 30: 933-943, 1982; Kurjan et al. , U.S. Patent No.
  • the PH05 signal sequence (Beck et al., WO 86/00637), the BAR1 secretory signal sequence (MacKay et al., U.S. Patent No. 4,613,572; MacKay, WO 87/002670; Welch et al., U.S. Patent No. 5,037,743), the SUC2 signal sequence (Carlson et al., Mol. Cell. Biol. 3 : 439-447, 1983).
  • preferred signal sequences include the ⁇ - 1-antitrypsin signal sequence (Kurachi et al., Proc. Natl. Acad. Sci.
  • ⁇ -2 plasmin inhibitor signal sequence Tone et al., J. Biochem. (Tokyo) 102: 1033-1042, 1987
  • tissue plasminogen activator leader sequence Pennica et al., Nature 301: 214-221, 1983.
  • a secretory signal sequence may be synthesized according to the rules established, for example, by von Heinje (Eur. J. Biochem. 133: 17-21, 1983; J. Mol. Biol. 184: 99-105, 1985; Nuc. Acids Res. 14: 4683-4690, 1986) .
  • Signal sequences may be used singly or may be combined.
  • a first signal sequence may be used singly or in combination with a sequence encoding the third domain of Barrier (described in U.S. Patent 5,037,743, which is incorporated by reference herein in its entirety) .
  • the third domain of Barrier may be positioned in proper reading frame 3' of the DNA sequence of interest or 5' to the DNA sequence and in proper reading frame with both the signal sequence and the DNA sequence of interest.
  • Host cells containing DNA constructs of the present invention are then cultured to produce GFAT.
  • the cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the particular host cell employed.
  • suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.
  • the growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker present on the DNA construct or co- transfected with the DNA construct.
  • Bacterial cells are preferably cultured in a complex, chemically undefined, medium comprising a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and in appropriate cases an antibiotic for the selection of plasmid-containing cells.
  • Bacterial cells transformed with expression units driven by inducible promoters are preferably cultured in a chemically defined medium that is either supplemented with the inducing substance or is lacking a nutrient whose deficiency induces the promoter.
  • bacterial cells transformed with an expression unit driven by the inducible tac promoter are preferably cultured in complex medium with the addition of isopropyl / 5-D-thiogalactopyranoside (IPTG) to induce expression of the desired protein from the promoter.
  • IPTG isopropyl / 5-D-thiogalactopyranoside
  • Yeast cells are preferably cultured in a chemically defined medium, comprising a carbon source, a non-amino acid nitrogen source, inorganic salts, vitamins and essential amino acid supplements.
  • the pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably at pH 6.5.
  • Methods for maintaining a stable pH include buffering and constant pH control, preferably through the addition of sodium hydroxide.
  • Preferred buffering agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, MO).
  • Yeast cells having a defect in a gene required for asparagine-linked glycosylation are preferably grown in a medium containing an osmotic stabilizer.
  • a preferred osmotic stabilizer is sorbitol supplemented into the medium at a concentration between 0.1 M and 1.5 M, preferably at 0.5 M or 1.0 M.
  • Cultured mammalian cells are generally cultured in commercially available serum- containing or serum-free media. Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art.
  • the GFAT produced according to the present invention may be purified by affinity chromatography on an antibody column using antibodies directed against GFAT.
  • Antibodies generated against GFAT may also be useful in detecting the presence of GFAT in cell lysates in immunological assays (reviewed by, for example, Sambrook et al., ibid.; which is incorporated by reference herein in its entirety) such as enzyme-linked immunosorbant assays and Western blot assays (Towbin et al. Proc. Natl. Acad. Sci. USA 7 : 4350, 1979).
  • the antibodies prepared against GFAT are capable of specifically binding to GFAT by which is meant that the antibodies against GFAT react with epitopes that are specific for GFAT.
  • Antibodies directed against GFAT or portions of GFAT may be generated using conventional techniques.
  • Portions of GFAT for use as immunogens may be chemically synthesized, such -I*- as by the solid-phase method of Barany and Merrifield (in The Peptides Vol. 2A, Gross and Meienhofer, eds, Academic Press, NY, pp. 1-284, 1979) or by use of an automated peptide synthesizer. Additional purification may be achieved by conventional chemical purification means, such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others. Methods of protein purification are known in the art (see generally, Scopes, R. , Protein Purification. Springer-Verlag, NY .
  • GFAT GFAT (1982), which is incorporated herein by reference) and may be ' applied to the purification of the recombinant GFAT described herein.
  • Substantially pure recombinant GFAT of at least about 50% is preferred, at least about 70-80% more preferred, and 95-99% or more homogeneity most preferred.
  • the present invention provides for the production of human GFAT essentially free of other proteins of human origin.
  • GFAT or portions thereof may be synthesized following any suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution addition.
  • Synthetic GFAT or portions thereof of the present invention may be prepared by hand synthesis or using a suitable peptide synthesizer such as an Applied Biosystems (Foster City, CA) Model 431A peptide synthesizer or the like.
  • recombinant human GFAT is used in assays to detect compounds capable of inhibiting GFAT activity.
  • Conditions and times sufficient for the inhibition of GFAT activity will vary with the particular assay used; however, conditions suitable for inhibition will generally be between 4°C and 55°C, preferably between 30°C and 40°C, under physiological conditions.
  • physiological conditions indicates conditions approximating the normal environment of cell- associated GFAT, and includes cell culture media and buffered, low-salt solutions within a pH range of between 5 and 9, preferably between 6.8 and 8.0.
  • Sufficient time for the inhibition and response will be between 5 and 60 minutes after exposure, with 15-30 minutes being particularly preferred. However, sufficient time will also be dependent on parameters of the assay such as protein concentrations and substrate concentrations.
  • Sufficient time for the inhibition of GFAT activity may be determined by varying a particular parameter such as substrate concentration in the assay and stopping the assay at specific time points. A plot of the varied parameter versus the assay time in assays containing no inhibitors may then be prepared and a time sufficient for inhibition may be chosen from the linear portion of the graph.
  • Recombinant GFAT for use within the assays of the present invention may be purified according to methods well known in the literature, may be partially purified or may be utilized within crude cell extracts. It may be preferable to purify the recombinant GFAT for use within the disclosed assays. Purification steps include ion exchange chromatography on Q fast flow, hydrophobic chromatography on organomercurial agarose and gel filtration. Purification of GFAT has been described, for example, by Dutka-Malen et al. (Biochimie 70: 287- 290, 1988) and Badet et al. (Biochemistry 26: 1940-1948, 1987) . *1
  • Glutamine:fructose-6-phosphate amidotransferase catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and glutamine.
  • Glutamate is a by ⁇ product of the GFAT-catalyzed reaction.
  • Assays for measuring GFAT activity generally rely on the measurement of the production of glucosamine-6-phosphate or the production of glutamate.
  • Glucosamine-6-phosphate production may be measured by the assay essentially described by Ghosh et al. (J. Biol. Chem. 255: 1265, I960,- which is incorporated by reference herein in its entirety) and Zalkin et al. (Meth. Enzymol.
  • a 96-well microtiter plate between 1 ng and 1 ⁇ g of human GFAT, preferably approximately between 0.5 and 1 ⁇ g of human GFAT, is inoculated into each well of a 96-well microtiter plate. Ethyl acetate-extracted test substances are diluted to between approximately 0.1X and 0.5X, and the diluted test substances are added to each well.
  • Reaction mixtures are prepared such that each well contains between 10 ⁇ K and 20 mM fructose-6-phosphate, preferably 10 mM fructose-6-phosphate; 0.25 ⁇ ci 3 H- fructose-6-phosphate; 10 mM L-glutamine; 30 mM sodium phosphate buffer (pH 7.5); 1 mM EDTA and 1 mM DTT.
  • the plate is vortexed and incubated at 37°C for a time sufficient to allow 3 H-glucosamine-6-phosphate production, preferably between 10 minutes and 1 hour. After incubation, each reaction is stopped by the addition of an equal volume of 1.0 M sodium borate (pH 8.5) .
  • the 3 H-glucosamine-6-phosphate content of each sample is assayed by cross-linking the 3 H-glucosamine-6- phosphate to a solid phase support that can be removed by filtration and counted by scintillation.
  • a solid phase support is a nylon powder (25,000, 3/32 inch diameter polished or unpolished nylon beads; The Hoover Group,. • Sault St. Marie, MI) that has been treated with hexane diamine (Aldrich Chemical) in the presence of triethyloxonium tetrafluoroborate (Aldrich Chemical) essentially as described by Van Ness et al. (Nuc. Acids Res. 10: 3345-3349, 1991).
  • nylon beads 25,000, 3/32 inch diameter polished or unpolished nylon beads
  • Triethyloxonium tetrafluoroborate is added to the mixture, which is stirred for an additional thirty minutes at room temperature.
  • the liquid is decanted, and the beads are quickly washed four times with l-methyl-2- pyrrolidinone.
  • the beads are stirred for twelve to twenty-four hours in 80% l-methyl-2-pyrrolidinone, 20% hexane diamine, after which the solution is decanted, and the beads are washed with l-methyl-2-pyrrolidinone follwoed by copious amounts of water.
  • the beads are dried in vacuo for four to five hours.
  • the amino groups on the derivatized powder are activated by the addition of a 10-fold excess of cyanuric chloride (Fluka, Buchs, altogether) prepared at 150 mg/ml in acetonitrile for thirty minutes at room temperature followed by multiple washes with 0.5 M borate buffer.
  • the derivatized nylon powder is added to the test reactions, and the primary amines present in the reaction mixture, including those on 3 H-glucosamine-6- phosphate quantitatively crosslink with the activated nylon powder.
  • the bound 3 H-glucosamine-6-phosphate is recovered by filtration on glass fiber mats (Pharmacia) , and the filters are counted on a Packard ⁇ -plate scintillation counter. A reduction in 3 H-glucosamine-6- phosphate production in the presence of a test sample relative to the 3 H-glucosamine-6-phosphate produced in the absence of the test substance indicates that the substance is a GFAT inhibitor.
  • recombinant GFAT preparations contain glucose-6-phosphate isomerase such as in cell extracts or in partially purified enzyme preparations
  • it will be necessary to completely remove the glucose-6-phosphate isomerase because it will rapidly deplete the 3 H-fructose-6-phosphate in the assay.
  • the recombinant GFAT may be purified away from the isomerase using a single-step p- hydroxymercuribenzoate affinity chromatography method (described by Hosoi et al. (Biochem. Biophys. Res. Comm. 85: 558-563, 1978; which is incorporated by reference herein in its entirety) wherein the recombinant GFAT is desorbed from the column matrix by elution with 20 mM DTT.
  • GFAT inhibitors are detected through their ability to reduce the conversion of radiolabeled glutamine to the byproduct radiolabeled glutamate.
  • GFAT inhibitors " are detected through their ability to reduce the conversion of glutamine to glutamate as measured by the reduction of 3-acteylpyridine adenine dinucleotide by glutamate dehydrogenase essentially as described by Traxinger and Marshall (J. Biol. Chem. 266: 10148-10154, 1991).
  • GFAT inhibitors are detected through their ability to reduce the conversion of 3 H-fructose-6- phosphate to 3 H-glucosamine-6-phosphate.
  • Inhibitors of GFAT activity are those substances that inhibit between 90% and 20% of GFAT activity levels, preferably between 80% and 50% of GFAT activity levels, relative to GFAT activity in the absence of the inhibitor.
  • a preferred GFAT inhibitor is a substance that provides between 90% and 20% inhibition, ' preferably between 80% and 50% inhibition of GFAT activity and does not confer any adverse physiological side effects.
  • Inhibitors of GFAT are administered at a level that results in a reduced level of GFAT activity and a concomitant increase in glucose utilization without any adverse physiological side effects.
  • compositions for parenteral and/or oral administration i.e., intravenously, subcutaneously, or intramuscularly
  • Compositions of GFAT inhibitors for parenteral administration generally comprise a solution of the inhibitor dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, 20-30% glycerol and the like.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting Jf agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • concentration of GFAT inhibitor in these formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • RNA extraction buffer 4 M guanidinium thiocyanate, 0.1 M Tris-HCl (pH 7.5), 1% b-mercaptoethanol, 0.5% sodium lauryl sarcosinate.
  • the fragmented tissue was homogenized for 20 seconds using a tissue homogenizer. Phenol:chloroformrisoamyl alcohol (50:48:2) was added, and the mixture was vortexed and centrifuged. The RNA was precipitated with isopropanol.
  • RNA was enriched using oligo d(T)-cellulose column chromatography as described by Sambrook et al. , eds. (Molecular Cloning: ⁇ 1
  • First strand cDNA was synthesized from the poly(A)+ RNA by first incubating 1.0 ⁇ g of the poly(A)+ RNA at 65°C for 3 minutes in 5 mM Tris-HCl (pH 7.0), 0.05 mM EDTA.
  • RNA was cooled on ice, and the cDNA synthesis reaction was primed with 5 pmol of oligonucleotide ZC2487 (Sequence ID NO: 3) in a 10 ⁇ l reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM each deoxynucleotide triphosphate, and 200 units of MMLV (RNase H " ) reverse transcriptase (GIBCO-BRL; Gaithersburg, MD) .
  • the reaction mixture was incubated at 45°C for 1 hour. After the incubation the mixture was diluted with 180 ⁇ l of 10 mM Tris-HCl (pH 7.6), 1 mM EDTA and stored at 4°C.
  • GFAT cDNA sequences were amplified from the first strand cDNA using degenerate oligonucleotide primers encoding GFAT DNA sequences by polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • Three microliters of the cDNA solution and 1 ⁇ M each of oligonucleotide ZC3866 and ZC3868 (Sequence ID NO: 4 and Sequence ID NO: 6) were combined in a reaction volume of 50 ⁇ l containing 200 ⁇ M of each deoxynucleotide triphosphate and IX Thermus aquaticus (Taq) buffer (Promega Corporation, Madison, WI) . The reaction was heated to 95°C for 5 minutes.
  • An aliquot of the amplification reaction was used for a second PCR reaction using different oligonucleotide primers, each of which contained a 5' tail of 10 nucleotides encoding convenient restriction enzyme sites for subcloning.
  • a one microliter aliquot of the first PCR reaction was combined with 1 ⁇ M each of the 3* oligonucleotide primers ZC3867 and ZC3869 (Sequence ID NO: 5 and Sequence ID NO: 7) in a final reaction volume of 50 ⁇ l that contained 200 ⁇ M of each deoxynucleotide triphosphate and IX Taq buffer (Promega) .
  • the reaction mixture was heated to 95°C for 5 minutes, after which the mixture was allowed to cool, and 2.5 units of Taq polymerase were added. The mixture was then overlaid with mineral oil. The PCR reaction was run for 40 cycles (thirty seconds at 95°C, thirty seconds at 42°C and sixty seconds at 7.2°C) followed by a 10 minute incubation at 72°C.
  • a 0.3 kb PCR reaction product was isolated by agarose gel electrophoresis. The purified fragment was ligated into pCRlOOO (Invitrogen, San Diego, CA) and. electroporated into E. coli strain DH10B (GIBCO-BRL) using a Bio-Rad Electroporator (Bio-Rad Laboratories; Richmond, CA) at 400 ohms, 25 ⁇ farads and 2000 volts. Aliquots of the transformed cells were plated onto LB plates containing 50 mg/1 Kanamycin (Sigma Chemical Co.; St. Louis, MO) .
  • a cDNA coding for a portion of human GFAT was obtained from a ⁇ gtll cDNA library prepared from HepG2 cells as described by Hagen et al. (Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986) .
  • the library was screened for sequences corresponding to the human GFAT DNA using clone 7-1 (described previously) . Approximately one million phage plaques were screened, and one positive plaque was identified.
  • the phage plaques were fixed to nylon filters (ICN Biomedicals, Inc. ; Irvine, CA) by placing 10 cm filters onto petri plates that had been plated with the library. The filters were removed and placed in lysing solution (0.5 M NaOH and 1.5. M NaCl) . After ' five minutes, the filters were neutralized for five minutes in 0.5 M Tris-HCl (pH 8.0), 1.5 M NaCl. ' After neutralization, the filters were rinsed in 2X SSPE (0.36 M NaCl, 20 mM NaH 2 P04 (pH 7.4), and 2 mM EDTA (pH 7.4) for one minute. The filters were allowed to air dry and were then baked in a vacuum oven at 80°C for 2 hours.
  • the PCR-generated DNA from clone 7-1 was used to prepare a probe using the MULTIPRIME Labeling Kit (Amersham; Arlington Heights, IL) according to the manufacturer's specifications.
  • the filters were prehybridized in 50% formamide/Ullrich's buffer (described above) at 37°C for approximately 18 hours, then hybridized for approximately 24 hours at 37°C in 50% formamide/Ullrich's buffer containing the radiolabeled probe. After hybridization, the filters were washed twice for 20 minutes each in a solution of 2X SSC, 0.1% sodium lauryl sarcosinate (SDS) at room temperature. A third wash was carried out in a solution of 0.5X SSC, 0.1% SDS at 50°C for 40 minutes. The filters were air dried, then exposed to X-ray film.
  • SDS sodium lauryl sarcosinate
  • a plug of agar corresponding to a positive area on the autoradiograph was picked from the master plate into 500 ⁇ l TM solution (10 mM Tris-HCl (pH 7.4), 10 mM MgCl2) and 20 ⁇ l chloroform to release the phage.
  • This isolate was designated 6-1.
  • the phage were plaque- ' purified by replating on E. coli strain Y1090 cells.
  • the filters were prepared, processed and probed as described above using the radiolabeled 7-1 PCR probe. The filters were washed twice in a solution of 2X SSC, 0.1% SDS at room temperature for 30 minutes. The filters were washed at 55°C and 65°C consecutively for 30 minutes each in 0.5X SSC, 0.1% SDS.
  • TM solution (10 mM Tris-HCl (pH 7.4); 10 mM MgCl2) and 20 ⁇ l chloroform, and the released phage were replated on to a lawn of Y1090 cells.
  • a plate lysate was prepared from each clone to obtain a phage titer.
  • Y1090 cells were grown in NZY media (Sambrook et al. , ibid.) to an OD600 of 5.6 and then infected with 5 x 106 plaque forming units (pfu) of phage.
  • the cultures were shaken at 37°C for 5.5 hours.
  • the cultures were centrifuged at 5,000 rpm for 10 minutes in a Sorvall GSA rotor (DuPont Co.; Wilmington, DE) .
  • the supernatants were collected, and 460 ⁇ g of RNa ⁇ e A (Sigma) and 460 ⁇ g of DNase I (Sigma) were added to each supernatant sample. After a S3
  • the pooled DNA was digested with Eco Rl, and a 1.1 kb fragment was gel purified.
  • the 1.1 kb DNA fragment and Eco Rl-linearized pUC19 were ligated.
  • the ligation mixture was used to electroporate E. coli strain DH10B cells.
  • the electroporated E. coli were plated on LB plates containing 50 mg/1 ampicillin.
  • the presence of insert in selected transformants was determined by PCR amplification using oligonucleotides ZC4306 and ZC4307 (Sequence ID NO: 8 and Sequence ID NO: 9) .
  • the selected transformants were inoculated into separate reaction mixtures each of which contained 200 ⁇ M of each deoxynucleotide triphosphate and IX Taq buffer in a final volume of 50 ⁇ l.
  • the reaction mixtures were heated to 95°C for 5 minutes.
  • the mixtures were allowed to cool and 2.5 units of Taq polymerase were added, and the mixtures were overlaid with mineral oil.
  • the PCR reactions were run for 30 cycles (thirty seconds at 95°C, thirty seconds at 50°C and sixty seconds at 72°C) followed by a 5 minute incubation at 72°C.
  • the PCR products were subjected to agarose gel electrophoresis. Transformants containing plasmids with insert exhibited 1.1 kb PCR reaction products.
  • Plasmid DNA was prepared from a transformant containing a plasmid with 1.1 kb insert. Sequence *1 analysis confirmed the insert size of 1.1 kb; however, the sequence did not extend to the initiation ATG.
  • plaque purification was carried out as previously described using Starks buffer (5X SSC, 25 mM sodium phosphate (pH 6.5), IX Denhardt's, 0.3 mg/ml salmon sperm DNA, 50% formamide, 10% dextran sulfate) in place of the Ullrich's hybridization buffer. Phage DNA was isolated from each clone as described above.
  • the GFAT insert from each clone was excised by Eco Rl digestion and gel purified as an approximately 3.1 kb fragment.
  • the insert DNA was sequenced and was found to encode GFAT.
  • a ⁇ clone containing a 3.1 kb GFAT sequence was designated 13.2.3.
  • Plasmid pBS(+) (Stratagene Cloning Systems; La
  • Jolla, CA was digested with Eco Rl and treated with calf alkaline phosphatase to prevent recircularization.
  • the phosphatased plasmid was gel purified and used in a ligation reaction with the Eco Rl-digested GFAT insert from ⁇ clone 13.2.3.
  • the ligation mixture was electroporated into E_. coli strain DH10B, and the presence of insert in selected transformants was determined by PCR as described previously. Plasmid DNA was prepared from a positive transformant, and the DNA was analyzed by digestion with Bam HI and Eco Rl to confirm the presence of the correct insert.
  • Plasmid pBSGFAT #1 was deposited on March 25, 1992 with the American Type Culture Collection (12301 Parklawn Dr., Rockville, MD) as an E. coli transformant under Accession No. 68946.
  • Human genomic GFAT clones were obtained from a human lung fibrobla ⁇ t library (Stratagene Cloning Systems, lambda FIX II #944201) . Approximately one million phage were adsorbed and plated with E. coli. strain LE392 (Stratagene Cloning Systems) . Plaques were lifted onto 1.2 ⁇ m BIOTRANS Nylon membranes (ICN Biomedicals, Inc.). The membranes were soaked for five minutes on filter paper saturated with Denaturing Buffer (0.5 N NaOH, 0.6 N NaCl). After denaturation, the filters were subjected to two sequential five-minute incubations in Neutralization Buffer (1.0 M Tris (pH 7.0), 1.5 M NaCl).
  • Neutralization Buffer 1.0 M Tris (pH 7.0), 1.5 M NaCl
  • the membranes were rinsed in a solution of 2x SSC, 0.1% SDS for ten minutes. After the rinse, the filters were blotted dry and baked for two hours at 80°C in vacuo. The membranes were prewashed at 42°C for one hour in 500 ml of 50 mM Tris (pH 8.0), 1 M NaCl, 1 mM EDTA, 0.1% SDS. After prewashing, the membranes were prehybridized in approximately 60 ml of Starks buffer containing 0.1% SDS for three to four hours at 42°C. A radiolabelled probe was prepared by first digesting plasmid pBSGFAT with Eco Rl and Pvu I.
  • the digested DNA was subjected to agarose gel electrophoresis, and the 3.1 kb GFAT cDNA fragment was purified using GENE CLEAN (Bio 101; La Jolla, CA) . After purification, the Eco Rl fragment was radiolabelled using the MEGAPRIME Kit (Amersham) .
  • the prehybridized membranes were hybridized in approximately 60 ml of Hybridization solution (Starks buffer containing 10% Dextran Sulfate, 0.1% SDS and approximately 4 X 10 7 CPM of the GFAT probe) overnignt at 42°C-i
  • Hybridization solution Startks buffer containing 10% Dextran Sulfate, 0.1% SDS and approximately 4 X 10 7 CPM of the GFAT probe
  • the membranes were subjected to a first wash in 2x SSC, 0.1% SDS for twenty minutes at room temperature followed by two sequential twenty-minute washes at 55°C in O.lx SSC, 0.1% SDS.
  • the membranes were autoradiographed. Six potential positive plaques were identified. Plugs of plaques encompassing the six potential positives were picked and adsorbed and plated with E.
  • plaques were picked into tubes containing 300 ⁇ l of adsorption buffer (10 mM MgCl 2 ,10 mM CaCl 2 ) , 200 ⁇ l of an exponential culture of E. coli strain LE392, and L broth containing 0.4%-maltose.
  • the cultures were grown for 10 minutes at 37°C, after which 10 ml of L broth containing 10 mM MgCl 2 and 0.1% glucose was added to each tube.
  • the tubes were shaken overnight at a 45° incline with the caps in the half-open position. After the overnight incubation, the tubes were centrifuged at 2000 rpm for 10 minutes.
  • the supernatants were decanted and centrifuged in a SW41 rotor (Beckman) at 30,000 rpm for 30 minutes. The supernatants were discarded, and the phage pellets were suspended in 200 ⁇ l of SM (Maniatis et al. , eds., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, 1982) .
  • the phage suspensions were each transferred to a microfuge tube and 200 ⁇ l of a freshly made 1 mg/ml proteinase K (Boehringer Mannheim) solution was added to each tube. The tubes were then incubated for 2 hours at 37°C. The suspensions were extracted once with phenol and once with chloroform.
  • the DNA was precipitated from the aqueous layer with 100 ⁇ l of 7.5 M ammonium acetate and 1 ml of 100% ethanol. The precipitates were pelleted by centrifugation, washed with ethanol and dried. The dried pellets were each resuspended in 100 ⁇ l of TE.
  • the DNA from clone la produced an approximately 14 kb insert upon digestion with Sal I.
  • the 14 kb fragment was gel purified and cloned into the Sal I site of pBS(+) (Stratagene). Two transformant clones containing the 14 kb insert in the pBS vector were designated pBSGFAT-la(#2) and pBSGFAT- la(#3).
  • the DNA from clone 15a was digested with Sal I and found to contain an approximately 20 kb insert.
  • Clone 15 a was digested with Xba I to produce two unique fragments of approximately 15 kb and 5 kb in size.
  • the 15 kb and 5 kb Xba I fragments were each gel purified and subcloned into Xba I-linearized pBS(+) (Stratagene Cloning Systems) .
  • Transformants containing the 15 kb inserts in the pBS(+) vector were designated pBSGFAT- 15aXl(#17) pBSGFAT-15aXl(#18) .
  • Transformants containing the 5 kb insert in the pBS(+) vector were designated pBSGFAT-15aX2(#25) and pBSGFAT-15aX2(#26) .
  • Restriction analysis of plasmids pBSGFAT-15aXl(#17) , pBSGFAT- 15aXl(#18), pBSGFAT-15aX2(#25) , pBSGFAT-15aX2(#26) , pBSGFAT-la(#2) and pBSGFAT-la(#3) suggested an overlap between clones la and 15a. All of the cDNA sequence lies within the subclone pBSGFAT-la#2.
  • the 2.0 kb fragment comprising the GFAT coding sequence was generated by PCR amplification using the 3.1 kb Eco Rl fragment from ⁇ clone 13.2.3 as a template.
  • Oligonucleotide ZC4839 (Sequence ID NO: 12) was designed to encode the first 19 base pairs of the GFAT coding t sequence and included a 5' terminal Eco Rl site.
  • Oligonucleotide ZC4866 (Sequence ID NO: 13) was designed to anneal to the 19 3 • terminal base pairs of the GFAT coding sequence and contained a second stop codon downstream of the coding sequence and a terminal Eco Rl site on the 3' end of the primer.
  • a 100 ⁇ l PCR reaction mixture was prepared containing 100 pmoles of each primer, 50 ng of the ⁇ clone 13.2.3 template, lx Taq Buffer (Promega), 200 ⁇ M dNTP's, and 5 units of Taq polymerase (Promega) .
  • the reaction mixture was amplified for 20 cycles (thirty seconds at 94°C, sixty seconds at 45°C and three minutes at 72°C) .
  • An aliquot of the PCR reaction was electrophoresed on an agarose gel, and a 2.0 kb fragment was isolated with GENE CLEAN (Bio 101) according to the manufacturer's directions. The 2.0 kb fragment was digested with Eco Rl and was ethanol was precipitated for 10 minutes on ice.
  • Plasmid pPROK-1 (Clontech Laboratories, Inc.; Palo Alto, CA) was digested with Eco Rl, treated with calf alkaline phosphatase to prevent recircularization , and gel purified with GENE CLEAN (Bio 101). The 2.0 kb Eco Rl fragment and the Eco RI- linearized pPROK-1 were ligated, ethanol precipitated, and the precipitated DNA was transfected into E. coli strain DH10B by electroporation.
  • insert in selected transformants was determined by PCR amplification as described above using oligonucleotides ZC4804 and ZC4307 (Sequence ID NO: 11 and Sequence ID NO: 9) , which were designed to hybridize to internal GFAT sequences.
  • Plasmid DNA was prepared from transformants containing insert, and the DNA was digested with Eco Rl and Sma I to determine which plasmids contained the insert in the proper orientation.
  • a plasmid containing the GFAT coding sequence in the proper orientation relative to the tac promoter in the vector pPROK-1 was digested with Eco Rl to isolate the 2.0 kb fragment encoding human GFAT.
  • Plamsid ZMB-3 is a derivative of the expression vector Zem228R.
  • Plasmid Zem228 is a pUC18-based expression vector containing a unique Bam HI site for insertion of cloned DNA between the mouse metallothionein-1 (MT-1) promoter and SV40 transcription terminator and an expression unit containing the SV40 early promoter, mouse neomycin resistance gene, and SV40 terminator.
  • Zem228 was modified to delete the two Eco Rl sites by partial digestion with Eco Rl, blunting with DNA polymerase I (Klenow fragment) and dNTPs, and re- ligation. Digestion of the resulting plasmid with Bam HI followed by ligation of the linearized plasmid with Bam HI-Eco Rl adapters resulted in a unique Eco Rl cloning site.
  • the resultant plasmid was designated Zem228R.
  • Plasmid ZMB-3 is similar to Zem228R but contains the adenovirus 2 major late promoter, adenovirus 2 tripartite leader, and 5* and 3' splice sites substituted for the MT-1 promoter.
  • the ligation mixture was electroporated into E. coli strain DH10B cells, and selected transformants were screened for the presence the insert as described above.
  • a plasmid containing the 2.0 kb insert was obtained and designated pZMBGFAT.
  • the 5' portion of the GFAT cDNA was altered to insert an Eco Rl site immediately upstream of the translation initiation codon.
  • Plasmid pBSGFAT was used as a template for PCR reaction which generated an approximately 0.7 kb fragment encoding the 5' portion of the GFAT cDNA.
  • Plasmid pBSGFAT was digested with Eco Rl to obtain the approximately 3.1 kb GFAT cDNA.
  • Oligonucleotide ZC6089 (Sequence ID NO: 18) was designed to encode a 5' Eco Rl restriction site immediately preceding the first 16 nucleotides of the GFAT coding sequence.
  • Oligonucleotide ZC6090 (Sequence ID NO: 19) was designed to anneal to GFAT coding sequence from nucleotide 801 to nucleotide 823 of Sequence ID NO: 1.
  • a 100 ⁇ l PCR reaction mixture was prepared containing 100 pmoles of each primer, 50 ng of the 3.1 kb Eco Rl fragment from pBSGFAT, lx Taq Buffer (Perkin Elmer Cetus) , 200 ⁇ M dNTP's, and 5 units of Taq polymerase (Perkin Elmer Cetus) .
  • the reaction mixture was amplified for five cycles (thirty seconds at 94°C, sixty seconds at 45°C and sixty seconds at 72°C) followed by 20 cycles (thirty seconds at 94°C, sixty seconds at 58°C and sixty seconds at 72°C) .
  • An aliquot of the PCR reaction was electrophoresed on an agarose gel, and an approximately 0.7 kb fragment was isolated with GENE CLEAN (Bio 101) according to the manufacturer's directions.
  • the gel- purified PCR fragment was digested with Eco Rl and Sma I to isolate the approximately 0.6 kb fragment.
  • the 3* GFAT coding sequence was obtained as a 1.4 kb Sma I-Eco Rl fragment from plasmid pZMBGFAT.
  • the Eco Rl-Sma I PCR fragment and the Sma I-Eco Rl fragment from plasmid pZMBGFAT were ligated into Eco Rl linearized pPROK-1 (Clontech) .
  • the ligation mixture was electroporated into E. coli strain DH10B cells, and selected transformants were screened for the presence the insert as described above. Plasmid containing the 2.0 kb insert were obtained and were designated pPROKGFAT #18 and pPROKGFAT #25.
  • the sequence of the cDNA in each plasmid was confirmed by sequence analysis.
  • Plasmid pPROKGFAT #18 was digested with Eco Rl, and the approximately 2.0 kb fragment containing the GFAT coding sequence was isolated by gel purification.
  • the 2.0 kb Eco Rl fragment was ligated with pZMB3 that had been linearized by digestion with Eco Rl and treated with calf alkaline phosphatase to prevent recircularization.
  • the ligation mixture was precipitated in the presence of approximately 20 ⁇ g of glycogen.
  • the i> precipitate was resuspended in water, and electroporated into E.
  • coli strain DH10B cells (10 kV, 200 ohms, 25 ⁇ F) . Transformants were selected in the presence of ampicillin. Clones containing the GFAT cDNA insert in the correct orientation relative to the promoter were identified by PCR amplification of portions of colonies. Portions of colonies of selected transformants were picked into 50 ⁇ l reaction mixtures containing 0.2 mM of each deoxyribonucleic acid, lx PCR buffer (Perkin Elmer Cetus) , 2 ⁇ M each of oligonucleotides ZC2435 and ZC5192 (Sequence ID NO: 16 and Sequence ID NO: 17, respectively) and 2.5 units of Taq polymerase (Perkin Elmer Cetus).
  • Oligonucleotide ZC2345 (Sequence ID NO: 16) is a sense primer corresponding to sequences in the Adenovirus major late promoter present in pZMB3, and oligonucleotide ZC5192 (Sequence ID NO: 17) is an antisense primer corresponding to sequences in the GFAT cDNA.
  • the PCR reactions were run for 30 cycles (forty-five seconds at 94°C, forty-five seconds at 58°C and forty-five seconds at 72°C) followed by a 5 minute incubation at 72°C. Aliquots of 10 ⁇ l were analyzed on a 1.5% agarose gel for the presence of an approximately 440 bp band indicating a correctly oriented cDNA insert. Plasmids pZMGFAT2-2 and pZMGFAT2-5 were identified as having the GFAT cDNA insert in the correct orientation relative to the promoter in pZMB3.
  • Plasmids pZMGFAT2-2 and pZMGFAT2-5 are transfected into suitable cultured mammalian cells using the Promega Transfecta Reagent (Promega Corp.; Madison, WI) according to the manufacturer-supplied directions. Stable clones of transfectants are selected in media containing G418. GFAT activity is assayed in selected transfectants.
  • Fifty microliters of an overnight culture of XL1-BLUE transformant containing pPROKGFAT #18 is i*. inoculated into 5 ml of LB-AMP (Sambrook et al., ibid.). The cultures are grown at 37°C until the Agoo of tne culture is approximately 0.5. Isopropyl J-D- thiogalactopyranoside (Sigma) is added to a final concentration of 10 mM to induce production of GFAT, and the culture is allowed to grow at 37°C for two hours. After induction, a 1 ml aliquot is removed from the culture, and the cells are pelleted.
  • the remainder of the culture is allowed to grow for 2 hours after which a 1 ml- aliquot is taken, and the cells are pelleted.
  • the pellets were either stored at -20°C before lysis or the pellets are immediately resuspended in 500 ⁇ l of lx sample buffer (0.07 M Tris-HCl (pH 6.8), 0.035% SDS, 10% glycerol, 0.1% bromphenol blue) and boiled for 10 minutes before loading a 25 ⁇ l aliquot onto a 10% SDS polyaerylamide gel.
  • the cell lysates are electrophoresed on a 10% SDS polyacrylamide gel, and the gel is stained with Coomassie blue to visualize the induced GFAT band.
  • the GFAT activity levels in pPROKGFAT #18 transformants are determined using the method essentially described by Richards and Greengard (Biochim. Biophvs Acta 304: 842-850, 1973; which is incorporated by reference herein) . Briefly, 50 ⁇ l of an overnight culture of XL1-BLUE transformant containing pPROKGFAT #18 is inoculated into 5 ml of LB-AMP (Sambrook et al., ibid.). The cultures are grown and induced as described above.
  • the cultures are split into five aliquots of 1 ml each.
  • the cells are pelleted, and the cell pellets are frozen at -20°C.
  • the cell pellets subjected to two freeze-thaw cycles, and each pellet is resuspended in 500 ⁇ l lysis buffer (100 ⁇ M PMSF, 100 mM NaH 2 P0 4 (pH 7.5), 50 mM KC1, 10 mM EDTA, 12 mM glucose 6- phosphate) .
  • the cells are lysed by sonication on ice.
  • the lysates are centrifuged in a microfuge, and the supernatants, representing the cytosol, are transferred to ice-cold tubes.
  • Total cytosolic protein is determined using a bicinchoninic acid protein assay kit (Pierce Chemical Co.; Rockford, IL) using the manufacturer's directions.
  • the supernatants are assayed for GFAT activity using the method essentially described by Richards and Greengard (supra.).
  • the reactions contain 20 ⁇ l of 100 mM fructose 6-phosphate (Sigma Chemical Co.), 20 ⁇ l of 100 mM glutamine (Sigma Chemical Co.), and 110 ⁇ l of a buffer containing 50 mM KC1 and 100 mM NaH 2 P0 (pH 7.5).
  • the reaction is initiated by the addition of 50 ⁇ l of cytosol, and the reaction is incubated for 37°C for one hour.
  • the reactions are terminated by the addition of 20 ⁇ l of a 50% (vol/vol) ice cold perchloric acid solution.
  • reaction mixtures are centrifuged for 10 minutes at 4°C in a microfuge.
  • the supernatants are transferred to fresh microfuge tubes, and 25 ⁇ l of ice cold 6 N KOH is added to each tube followed by centrifugation at 4°C for 10 minutes in a microfuge.
  • the glucosamine-6-phosphate content of the supernatant is determined using a modification of the colorimetric method of Levvy and McAllan (Biochemistry 73: 127-132, 1959; which is incorporated by reference herein) . Briefly, 150 ⁇ l of the supernatant from each sample is added to a 12 x 75 mm glass tubes followed by 100 ⁇ l of a saturated tetraborate solution and 10 ⁇ l of 1.75% (vol/vol) acetic anhydride in ice cold acetone is added. The reaction mixtures are briefly agitated and placed in a boiling water bath for 4 minutes. After heating, the reactions are placed in an ice water bath for one minute.
  • Glucosamine-6-phosphate is used as. a standard, and a unit of GFAT activity is defined as the amount of GFAT that catalyzes the formation of 1 nmole of glucosamine-6-phosphate/min at 37°C.
  • UDP-GlcNAc uridine 5'diphosphate n-acetylglucosamine
  • UPD-GlcNAc inhibits human and yeast GFAT, but does not affect the bacterial form of the enzyme.
  • UDP-GlcNAc sensitivity of GFAT activity is assessed in reaction mixtures that are identical with those described above with the exception that the buffer volume as reduced to 100 ⁇ l and 10 ⁇ l of a 5 mM solution of UDP-GlcNAc (Sigma Chemical Co., St. Louis, MO) was added.
  • the reactions are again initiated by the initiation of 50 ⁇ l of cytosol, and the reactions are allowed to proceed for one hour at 37°C.
  • the reactions are terminated, and the levels of glucosamine- 6-phosphate are determined as described above.
  • Antisera against recombinant GFAT and a GFAT peptide were raised in New Zealand white rabbits (R and R Rabbitry; Stanwood, CA) and in Balb/c mice (Simonsen Labs; Gilroy, CA) .
  • E. coli transformants containing either pPROK-1 or pPROGFAT were grown, induced and harvested as described above (Example 3) . Lysates corresponding to 1- 2 ml of cultured, induced cells were thawed, diluted in sample buffer (0.07 M Tris-HCl (pH 6.8), 0.035% SDS, 10% glycerol, 0.1% bromphenol blue) and electrophoresed on a preparative SDS-polyacrylamide gel.
  • the protein was transferred to nitrocellulose (Schleicher & Schuell; Keene, NH) in lx Transfer Buffer (Table 2) in a Bio-Rad TRANSBLOT (BioRad Laboratories; Richmond, CA) at 400 milliamps for ninety minutes at 4°C.
  • the nitrocellulose was rinsed in distilled water and stained for five minutes in Ponceau S (Sigma; 0.1% w/v in 1% acetic acid).
  • the nitrocellulose was destained in distilled water.
  • the 77 kDa GFAT band was identified and cut out with a clean razor blade.
  • the nitrocellulose band was destained completely in water and dried. This procedure generated enough protein for the immunization of five mice.
  • a synthetic peptide having the sequence of Sequence ID NO: 2 from Cysteine, amino acid number 493, to Glutamic acid, amino acid number 505, and containing a C-terminal Tyrosine residue to facilitate conjugation was synthesized on an Applied Biosystems Model 431A peptide synthesizer (Foster City, CA) using standard cycles as 11 directed by the manufacturer and Fmoc chemistry essentially as described by Carpino and Han (J. A er. Chem. Soc. 92: 5748-5749, 1970; J. Org. Chem. 37: 3404- 3409, 1972).
  • An unloaded HMP p-alkyloxybenzyl alcohol
  • the peptide was cleaved from the resin using 95% trifluoroacetic acid (TFA) .
  • TFA trifluoroacetic acid
  • the peptide was precipitated in diethyl ether and redissolved in 10% acetic acid.
  • the peptide was purified on a reverse-phase HPLC using a C-4 column with a H 2 0/acetonitrile (both containing 0.1% TFA) gradient. The main peak was collected, a sample was taken for amino acid analysis and the peptide was lyophilized.
  • the peptide was coupled to KLH activated maleimide (Chemicon; Temecula, CA) for use as an immunogen. Ten milligrams of the peptide was diluted in 400 ⁇ l of 10 mM HAc.
  • a reaction mixture containing the peptide, 5.6 ml phosphate buffered saline (PBS; Sigma Chemical Co., St. Louis, MO) and 1 ml of a 10 mg/ml KLH activated maleimide solution was rocked at 4°C for 22 hours.
  • the resulting mixture was aliquotted into 24 vials containing approximately 410 ⁇ g of conjugated peptide per vial.
  • Two rabbits were injected subcutaneously with 205 ⁇ g of conjugated peptide each every three weeks.
  • Five female Balb/C mice were injected intraperitoneally with 82 ⁇ g of conjugated peptide each every two weeks for a total of six injections.
  • the nitrocellulose band containing GFAT (prepared as described above) was dissolved in a minimal amount of DMSO using between 250 ⁇ l and 375 ⁇ l of DMSO, but not more than 500 ⁇ l of DMSO.
  • the DMSO-solubilized nitrocellulose bands were divided into five equal aliquots. Five young female Balb/c mice were injected intraperitoneally with the DMSO-solubilized nitrocellulose bands of GFAT. The injections were repeated every two weeks for a total of six injections.
  • E. coli strain XL1-BLUE (Stratagene) transformed with either pPROGFAT or pPROK-1 (negative control) were grown overnight at 37°C in LB media (Sambrook et al., ibid.) supplemented with 100 ⁇ g ampici11in/ml.
  • the cultures were diluted 1:40 into 5 ml of LB media, and the cultures were grown for 2.5 hours at 37°C.
  • the cells were pelleted, and the cell pellets were resuspended in 5 ml M9 media (Sambrook et al. , ibid.).
  • Each culture received IPTG to a final concentration of 10 mM, and the cultures were grown for 2 hours at 37°C. After the 2 hour period, 25 ⁇ Ci/ml of 35 S-EXPRESS (NEN; Boston, MA) was added to each culture, and the cultures were incubated for 10 minutes at 37°C. After incubation, the cells were centrifuged, and the cell pellets were washed twice with PBS. The volume of the cell pellets were approximated, and 10 volumes of a solution containing 50 mM glucose, 10 mM EDTA, 25 mM Tris (pH 8.0) and 4 mg/ml lysozyme was added to each pellet to lyse the cells.
  • the resuspended pellets were incubated for 5 minutes at room temperature after which 500 ⁇ l of lx RIPA (Table 2) was added to each lysate.
  • the lysates were incubated on ice for 30 minutes followed by centrifugation in a microfuge for 15 minutes at 4°C.
  • the supernatants were transferred to fresh tubes and were stored at -70°C.
  • the lysates were thawed on ice and then centrifuged in a microfuge for 15 minutes to obtain clarified supernatants.
  • a 30 ⁇ l aliquot of each clarified lysate was electrophoresed on an SDS-polyacrylamide gel, and the gel was exposed to film.
  • the lysates were then immunoprecipitated with the rabbit and mouse polyclonal antibodies.
  • 100 ⁇ l of lysate was combined with 100-fold diluted antisera of either the rabbit anti-peptide polyclonal, the mouse anti-peptide polyclonal or the mouse anti-recombinant GFAT polyclonal, and the reactions were incubated on ice for 60 minutes.
  • 5 ⁇ l of rabbit anti-mouse polyclonal antibodies Sigma was added to each reaction mixture, and the mixtures were incubated for 30 minutes on ice.
  • the boiled samples were subjected to SDS-polyacrylamide gel electrophoresis, and the gel was fixed in 10% HAc, 20% methanol for 30 minutes. After fixing, the gel was soaked in AMPLIFY (Amersham; Arlington Hts., IL) for 20 minutes. The gel was dried and exposed to film with a screen at -70°C. The presence of bands at 77 kDa indicated that each antisera was capable of immunoprecipitating recombinant human GFAT. Anti-GFAT peptide rabbit antisera is fO
  • affinity purified on a GFAT peptide column affinity purified on a GFAT peptide column. Affinity purified antisera is used in methods for purifying GFAT.
  • GTGTGCACCC CCGATCCCGC CAGCCACTCG CCCCTGGCCT CGCGGGCCGT GTCTCCGGCA 120
  • CAG AAA CTA GCA ACA GAA CTT TAT CAT CAG AAG TCA GTT CTG ATA ATG 1751 Gin Lys Leu Ala Thr Glu Leu Tyr His Gin Lys Ser Val Leu He Met 530 535 540

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Abstract

L'invention se rapporte à des molécules d'ADN isolées codant pour la glutamine:fructose-6-phosphate amidotransférase humaine. Ces molécules d'ADN sont utiles dans des procédés pour dépister des antagonistes de glutamine:fructose-6-phosphate amidotransférase. En résumé, ces molécules d'ADN codant pour la glutamine:fructose-6-phosphate amidotransférase humaine sont exprimées dans des cellules hôtes appropriées, et une glutamine:fructose-6-phosphate amidotransférase recombinée est produite. Une substance test est exposée à la glutamine:fructose-6-phosphate amidotransférase humaine recombinée en présence de fructose-6-phosphate et de glutamine. Une réduction de l'activité de la glutamine:fructose-6-phosphate amidotransférase, par rapport à son activité en l'absence de la substance test, indique la présence d'un composé qui inhibe la glutamine:fructose-6-phosphate amidotransférase humaine.
PCT/US1993/003773 1992-04-22 1993-04-22 Glutamine:fluctose-6-phosphate amidotransferase humaine WO1993021330A1 (fr)

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US5876713A (en) * 1996-08-13 1999-03-02 Takeda Chemical Industries Ltd Glutamine: fructose-6-phosphate amidotransferase, its production and use
WO2000037617A1 (fr) * 1998-12-22 2000-06-29 G.D. Searle & Co. Glutamine: une enzyme de type ii, la fructose-6-phosphate aminotransferase, et ses acides nucleiques codants
WO2001020327A1 (fr) * 1999-09-15 2001-03-22 Astrazeneca Ab Dosage de la scintillation par proximite dans un procede permettant l'identification de modulateurs de la glucokinase
WO2001096574A1 (fr) * 2000-06-13 2001-12-20 Otsuka Pharmaceutical Co., Ltd. Nouveau gene enzymatique et son produit d'expression
WO2003023063A1 (fr) * 2001-09-07 2003-03-20 Sankyo Company, Limited Methode d'estimation du risque d'apparition de diabetes
US6632627B2 (en) 1997-08-12 2003-10-14 Takeda Chemical Industries, Ltd. Glutamine: fructose-6-phosphate amidotransferase, its production and use
EP1431396A1 (fr) * 2002-12-19 2004-06-23 F. Hoffmann-La Roche Ag Procédé de mesure de glucosamine-6-phosphate
FR2857374A1 (fr) * 2003-07-08 2005-01-14 Centre Nat Rech Scient Glutamine:fructose-6-phosphate amidotransferase(gfat) comprenant une etiquette de purification interne, et son utilisation pour le criblage de composes
CN112980914A (zh) * 2019-12-12 2021-06-18 中国科学院大连化学物理研究所 Gfpt1基因作为靶点在筛选抗肿瘤药物中的应用

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BIOCHEMISTRY. vol. 26, 1987, EASTON, PA US pages 1940 - 1948 BADET,B. ET AL. 'Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location' cited in the application *
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 242, no. 13, 10 July 1967, BALTIMORE, MD US pages 3135 - 3141 KORNFELD, R. 'Studies on L-glutamine D-fructose 6-phosphate Amidotransferase' *
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 264, no. 15, 25 May 1989, BALTIMORE, MD US pages 8753 - 8758 WATZELE, G. ET TANNER, W. 'Cloning of the glutamine:fructose-6-phosphate amidotransferase gene from yeast' cited in the application *
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 266, no. 16, 5 June 1991, BALTIMORE, MD US pages 10148 - 10154 TRAXINGER, R.R. ET MARSHALL, S. 'Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine' cited in the application *
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 266, no. 16, 5 June 1991, BALTIMORE, MD US pages 10155 - 10161 MARSHALL, S. ET AL. 'Complete inhibition of glucose-induced desensitization of the glucose transport system by inhibitors of mRNAs synthesis' cited in the application *
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 267, no. 35, 15 December 1992, BALTIMORE, MD US pages 25208 - 25212 MCKNIGHT, G.L. ET AL. 'Molecular cloning, cDNA sequence, and bacterial expression of human glutamine;fructose-6-phosphate amidotransferase' *
THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY vol. 5, 1974, pages 515 - 523 TRUJILLO, J. L. & GAN, J.C. 'Purification and some kinetic properties of bovine thyroid gland L-glutamine : D-fructose-6-phosphate amidotransferase' *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6207431B1 (en) 1996-08-13 2001-03-27 Tekeda Chemical Industries, Ltd. Glutamine:fructose-6-phosphate amidotransferase, its production and use
EP0824149A3 (fr) * 1996-08-13 1999-07-21 Takeda Chemical Industries, Ltd. Glutamine: fructose-6-phosphate amidotransférase (GFAT), sa production et son utilisation
US5876713A (en) * 1996-08-13 1999-03-02 Takeda Chemical Industries Ltd Glutamine: fructose-6-phosphate amidotransferase, its production and use
US6632627B2 (en) 1997-08-12 2003-10-14 Takeda Chemical Industries, Ltd. Glutamine: fructose-6-phosphate amidotransferase, its production and use
WO2000037617A1 (fr) * 1998-12-22 2000-06-29 G.D. Searle & Co. Glutamine: une enzyme de type ii, la fructose-6-phosphate aminotransferase, et ses acides nucleiques codants
WO2001020327A1 (fr) * 1999-09-15 2001-03-22 Astrazeneca Ab Dosage de la scintillation par proximite dans un procede permettant l'identification de modulateurs de la glucokinase
US7091018B2 (en) 2000-06-13 2006-08-15 Otsuka Pharmaceutical Co., Ltd. Enzyme gene and its expression product
WO2001096574A1 (fr) * 2000-06-13 2001-12-20 Otsuka Pharmaceutical Co., Ltd. Nouveau gene enzymatique et son produit d'expression
JP2002065284A (ja) * 2000-06-13 2002-03-05 Otsuka Pharmaceut Co Ltd 新規酵素遺伝子およびその発現産物
CN100390286C (zh) * 2000-06-13 2008-05-28 大塚制药株式会社 新酶基因及其表达产物
WO2003023063A1 (fr) * 2001-09-07 2003-03-20 Sankyo Company, Limited Methode d'estimation du risque d'apparition de diabetes
EP1431396A1 (fr) * 2002-12-19 2004-06-23 F. Hoffmann-La Roche Ag Procédé de mesure de glucosamine-6-phosphate
FR2857374A1 (fr) * 2003-07-08 2005-01-14 Centre Nat Rech Scient Glutamine:fructose-6-phosphate amidotransferase(gfat) comprenant une etiquette de purification interne, et son utilisation pour le criblage de composes
JP2007515931A (ja) * 2003-07-08 2007-06-21 サントル・ナショナル・ドゥ・ラ・ルシェルシュ・シャンティフィク 精製用内部マーカーを含むグルタミン：フルクトース−６−リン酸アミドトランスフェラーゼ（ｇｆａｔ）および化合物のスクリーニングのためのその使用
WO2005005628A1 (fr) * 2003-07-08 2005-01-20 Centre National De La Recherche Scientifique Glutamine:fructose-6-phosphate amidotransferase (gfat) comprenant une etiquette de purification interne, et son utilisation pour le criblage de composes
US7625734B2 (en) 2003-07-08 2009-12-01 Centre National De La Recherche Scientifique (Cnrs) Glutamine:fructose-6-phosphate amidotransferase (GFAT) comprising an internal purification marker and use thereof for the screening of compounds
CN112980914A (zh) * 2019-12-12 2021-06-18 中国科学院大连化学物理研究所 Gfpt1基因作为靶点在筛选抗肿瘤药物中的应用

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