Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • 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
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/18—Baker's yeast; Brewer's yeast
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Saccharomyces cerevisiae erg4 mutants for the expression of glucose transporters from mammals.
• the invention relates to yeast strains in which the human Glut 4 and Glut 1 transporter can be functionally expressed.
• heterotrophic cells transport glucose into the interior of the cell via special transporter proteins.
• Different mechanisms that mediate glucose transport have developed in the various organisms, such as, in particular, proton support systems, Na + glucose transporters, binding protein-dependent systems, phosphotransferase systems and systems for facilitated diffusion.
• GLUT glucose transporter
• HXT hexose transporter
• Glucose transport plays a major role in diseases that are associated with defective glucose homeostasis, such as diabetes mellitus or Fanconi-Bickel syndrome.
• the transport of glucose in mammals has therefore been the subject of numerous studies.
• thirteen proteins similar to glucose transporters GLUT1 to GLUT12, HMIT - H-myo-inositol transporter
• the key roles of these transporters include the uptake of glucose in various tissues, their storage in the liver, their insulin-dependent uptake in muscle cells and adipocytes, and the measurement of glucose by the ß cells of the pancreas.
• GLUT1 mediates glucose transport into the erythrocytes and through the blood-brain barrier, but is also expressed in many other tissues, while GLUT4 is restricted to insulin-dependent tissues, primarily muscle and adipose tissue. In these insulin-dependent tissues, the control of the targeting of GLUT4 transporters in intracellular compartments or Plasma membrane compartments are an important mechanism for regulating glucose uptake. In the presence of insulin, intracellular GLUT4 is redistributed to the plasma membrane to facilitate glucose uptake. GLUT1 is also expressed in these insulin-dependent tissues, and its distribution in the cell is also affected by insulin, but less so. In addition, the relative effectiveness with which GLUT1 or GLUT4 catalyze sugar transport is determined not only by the extent of each transporter's targeting to the cell surface, but also by their kinetic properties.
• Yeast cells are unicellular eukaryotic organisms. For some proteins, they are therefore more suitable for expression than bacterial systems, in particular with regard to the implementation of screening assays for the identification of pharmaceutically active substances.
• the present invention relates to a purified and isolated polynucleotide comprising a DNA sequence which codes for the GLUT4V85M protein.
• This protein contains an amino acid exchange from valine to methionine at position 85 of the amino acid chain of the human GLUT4 protein.
• This changed GLUT4V85M protein opens up further alternatives for the expression of a functional GLUT4 protein.
• the glucose uptake can be determined either by transport measurements using radiolabelled glucose or by growth on medium with glucose as the only carbon source.
• the purified and isolated polynucleotide comprising a DNA sequence which codes for a protein GLUT4V85M can comprise or consist of a sequence of the following groups: a) a nucleotide sequence, according to Seq ID No. 1, b) a nucleotide sequence which hybridizes under stringent conditions to a sequence of Seq ID No. 1 and which codes for a protein GLUT4V85M.
• the purified and isolated polynucleotide preferably encodes a GLU4V85M protein which has an amino acid sequence of Seq ID No. 2.
• the purified and isolated polynucleotide comprising a DNA sequence which codes for a protein GLUT4V85M as described above can be operatively linked to a promoter.
• a promoter such as the Lac, trp, ADH or HXT7 promoter are suitable as promoters.
• the part of the polynucleotide coding for the protein GLUT4V85M is operatively connected to a promoter if and only by means of this promoter, with the aid of a vector, an mRNA is formed in a bacterial or eukaryotic organism which can be translated into the protein GLUT4V85M.
• Such a vector is, for example, the vector p4H7GLUT4V85M (Seq ID No. 3).
• the protein GLUT4V85M can be expressed in yeast cells using this vector.
• the polynucleotide described above, comprising a DNA sequence which codes for a protein GLUT4V85M, is suitable in a preferred embodiment for replicating this polynucleotide in a yeast cell or for expressing that for Protein GLUT4V85M coding part of the polynucleotide into the protein GLUT 4 V85M in a yeast cell.
• a yeast cell from Saccharomyces cerevisiae is particularly suitable.
• the polynucleotide For replication and expression in a yeast cell, the polynucleotide comprises a DNA sequence, which codes for a protein GLUT4V85M, as a yeast vector.
• the region of the polynucleotide coding for the protein GLUT4V85M can be operatively linked to a promoter specific for yeast cells, such as, for example, the ADH promoter (alcohol dehydrogenase promoter) or HXT7 promoter (hexose transporter promoter).
• the yeast vectors are a group of vectors that were developed for cloning DNA in yeast.
• a yeast cell is preferably a yeast cell deposited as Saccharomyces cerevisiae DSM 15187 as in the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 16, 38124 Braunschweig).
• the invention also relates to a yeast cell in which all glucose transporters are no longer functional and in which no functional Fgy1 protein and no functional Erg4 protein is contained.
• the absence of an Erg4 protein or an Fgy1 protein can in particular be due to an interruption of the coding genome sections in question or to partial or complete removal of the coding genome sections.
• a yeast cell which contains no functional glucose transporters, no functional Fgy1 protein and no functional Erg4 protein a yeast cell stored as Saccharomyces cerevisiae DSM 15184 as in the DSMZ is preferably used.
• a yeast cell as described above is preferably used to express a GLUT1 protein or a GLUT4 protein of a mammal, such as, in particular, rats, mice, rabbits, pigs, cattle or apes.
• the yeast cell is used to express a human GLUT4 or GLUT1 protein.
• a yeast cell from Saccharomyces cerevisiae, all of whose glucose transporters and the Erg4 protein are no longer functional, can contain a polynucleotide of this invention which comprises a DNA sequence which codes for a protein GLUT4V85M. This yeast cell can also express the protein GLUT4V85M and thus contain this protein.
• Such a yeast strain containing a polynucleotide, which comprises a DNA sequence coding for the protein GLUT4V85M is preferably the yeast strain Saccharomyces cerevisiae DSM 15185 deposited with the DSMZ.
• a yeast cell, all of which glucose transporters and the Erg4 protein are no longer functional, which are a polynucleotide comprising a DNA sequence encoding a protein GLUT4V85M can be produced, for example, by a) providing a yeast cell, all of whose glucose transporters and the Erg4 protein are no longer functional, b) an isolated and purified polynucleotide, which comprises a DNA sequence coding for the protein GLUT4V85M and can be replicated in a yeast cell, c) the yeast cell from a) is transformed with the polynucleotide from b), d) a transformed yeast cell is selected, e) optionally the protein GLUT4V85M is brought to expression.
• An isolated and purified polynucleotide which comprises a DNA sequence coding for the protein GLUT4V85M is preferably a vector which can be replicated in a yeast cell and in which the DNA sequence has been cloned.
• a vector is, for example, p4H7GLUT4V85M (Seq ID No. 3).
• the invention also relates to a yeast cell whose all glucose transporters and their proteins for Fgy1 and Erg4 are no longer functional and which contains a polynucleotide which comprises a DNA sequence coding for the protein GLUT4V85M.
• This yeast cell can also express the protein GLUT4V85M and thus contain this protein.
• a yeast strain preferably the yeast strain Saccharomyces cerevisiae DSM 15186 deposited with the DSMZ.
• a yeast cell whose all glucose transporters and the proteins Fgy1 and Erg4 are no longer functional and which contains a polynucleotide comprising a DNA sequence which codes for the protein GLUT4V85M can be produced, for example, by a) a yeast cell, all of which glucose transporters as well the proteins Fgy1 and Erg4 are no longer functional, b) an isolated and purified polynucleotide which comprises a DNA sequence coding for the protein GLUT4V85M and can be replicated in a yeast cell is provided, c) the yeast cell from a) is provided is transformed with the polynucleotide from b), d) a transformed yeast cell is selected, e) optionally the protein GLUT4V85M is brought to expression.
• the above-mentioned isolated and purified polynucleotide which comprises a DNA sequence coding for the protein GLUT4V85M, is preferably a vector which can be replicated in a yeast cell and in which the DNA sequence has been cloned.
• a vector is, for example, p4H7GLUT4V85M (Seq ID No. 3).
• the invention also relates to a yeast cell, the glucose transporters of which are no longer functional, which contains a polynucleotide comprising a DNA sequence which codes for the protein GLUT4V85M.
• This yeast cell can also express the protein GLUT4V85M and thus contain this protein.
• a yeast strain is preferably the yeast strain Saccharomyces cerevisiae 15188 deposited with the DSMZ.
• a yeast cell whose all glucose transporters are no longer functional and which contains a polynucleotide comprising a DNA sequence which codes for the protein GLUT4 V85M can be produced, for example, by a) a yeast cell, the glucose transporter of which is no longer functional, is provided, b) an isolated and purified polynucleotide is provided, which comprises a DNA sequence coding for the protein GLUT4V85M and can be replicated in a yeast cell, c) the yeast cell transforming from a) with the polynucleotide from b), d) selecting a transformed yeast cell, e) optionally expressing the protein GLUT4V85M.
• the above-mentioned isolated and purified polynucleotide which comprises a DNA sequence coding for the protein GLUT4V85M, is preferably a vector which can be replicated in a yeast cell and in which the DNA sequence has been cloned.
• a vector is, for example, p4H7GLUT4V85M (Seq ID No. 3).
• the invention also relates to a protein of the amino acid sequence according to Seq ID No. 2. This protein is a human GLUT4 protein in which a valine is replaced by a methionine at position 85 of the amino acid chain.
• the invention also relates to a method for identifying a compound which stimulates the activity of a GLUT4 protein, characterized in that a) a yeast cell is provided, all of which glucose transporters and the Erg4 protein are no longer functional, and which comprise a polynucleotide comprising a DNA Sequence encoding a protein GLUT4V85M contains, b) a chemical compound is provided, c) the yeast from a) is brought into contact with the chemical compound from b), d) the glucose uptake of the yeast from c) is determined, e) the determined value of the glucose uptake from d) is related to the ascertained value of the glucose uptake in a yeast cell according to a) which has not been brought into contact with a chemical compound according to b), a compound which is an increase in the ingested
• Amount of glucose in the yeast according to d) causes the activity of the GLUT4V85M protein to be stimulated.
• the activity of the GLUT4V85M Stimulate proteins is believed to also stimulate GLUT4 activity.
• the invention also relates to a medicament which contains a compound which has been identified by the method described above, and furthermore additives and auxiliaries for the formulation of a medicament.
• the invention further relates to the use of a compound which has been identified by the method described above for the manufacture of a medicament for the treatment of type I and / or II diabetes.
• the invention also relates to a medicament containing a compound which has been identified by the method described above, and additives and auxiliaries for formulating a medicament.
• the invention further relates to the use of a compound which has been identified by the method described above for the production of a medicament for the treatment of diabetes.
• the invention further relates to the use of a compound which has been identified by a method described above for the production of a medicament for the treatment of diabetes.
• the present invention also includes a method for identifying a compound which has been identified by a method described above for the production of a medicament for the treatment of diabetes.
• the present invention also includes a method for identifying a compound which has been identified by a method described above for the production of a medicament for the treatment of diabetes.
• the present invention also includes a method for identifying a
• Yeast cell can be replicated, b) a chemical compound is provided, c) the yeast from a) is brought into contact with the chemical compound from b), d) the glucose uptake of the yeast from c) is determined, e) the value determined Glucose uptake from d) is related to the determined value of glucose uptake in a yeast cell according to a), which is not brought into contact with a chemical compound according to b) was, wherein a compound that causes an increase in the amount of glucose in the yeast according to d) inhibits the activity of the protein Erg4.
• the invention further relates to a method for identifying a compound which inhibits the corresponding protein of the FGY1 gene, characterized in that a) a yeast cell is provided, the glucose transporters and the Erg4 protein of which are no longer functional and which contains a GLUT4 protein , is provided, b) a chemical compound is provided, c) the yeast from a) is brought into contact with the chemical compound from b), d) the glucose uptake of the yeast from c) is determined, e) the determined value of the glucose uptake from d) is related to the determined value of the glucose uptake in a yeast cell according to a) which has not been brought into contact with a chemical compound according to b), a compound which shows an increase in the amount of glucose taken up in the yeast according to d ) causes the activity of the protein Fgy1 to be inhibited.
• the invention also relates to a medicament containing a compound which was identified by the method described above, and additives and auxiliaries for the formulation of a medicament.
• Hybridization means the assembly of two nucleic acid single strands that have complementary base sequences into double strands. Hybridization can take place between two DNA strands, one DNA and one RNA strand, and between two RNA strands.
• hybrid molecules can be produced by heating the nucleic acids involved, which may initially be double-stranded, to such an extent that they become single-stranded molecules without Secondary structure decay. This is done, for example, by boiling in a water bath for 10 minutes. Then let them cool slowly. During the cooling phase, complementary chains are paired to form double-stranded hybrid molecules.
• Hybridizations are usually carried out under laboratory conditions with the aid of hybridization filters onto which single-stranded or denaturable polynucleotide molecules are applied by blotting or electrophoresis.
• the hybridization can be made visible with corresponding complementary polynucleotide molecules by providing these polynucleotide molecules to be hybridized with a radioactive fluorescent label.
• Stringency describes the degree of agreement or accuracy of fit of certain conditions. With high stringency, the requirements for agreement are higher than with low stringency.
• certain, differently stringent conditions are set depending on the application and the objective. In the case of high stringency, the reaction conditions in the hybridization are set such that only complementary molecules that match very well can hybridize with one another. Low stringency also enables partial hybridization of molecules with more or less large sections of unpaired or mismatched bases.
• hybridization conditions should be understood as stringent, in particular if the hybridization is carried out in an aqueous solution containing 2x SSC at 68 ° C. for at least 2 hours and then first for 5 minutes in 2x SSC / 0.1% SDS at room temperature, then for 1 hour in 1x SSC / 0.1% SDS at 68 ° C and for 1 additional hour in 0.2% SSC / 0.1% SDS at 68 ° C.
• a 2x SSC, 1x SSC or 0.2x SSC solution is prepared by diluting a 20x SSC solution.
• a 20x SSC solution contains 3 mol / l NaCl and 0.3 mol / l Na citrate.
• the pH is 7.0.
• the skilled worker is familiar with the methods for hybridizing polynucleotides under stringent conditions. He finds corresponding instructions in specialist books, in particular the Current Protocols in Molecular Biology (Wiley Interscience; editor: Frederich M. Ausubel, Roger Brant, Robert E. Scientific, David J. Moore, JG Seidmann, Kevin Struhl; ISBN: 0-471 -50338-X).
• Ylp vectors Yeast integrating plasmids
• yeast vectors essentially correspond to the vectors used for cloning in bacteria, but contain a selectable yeast gene (eg URA3, LEU2).
• plasmids are derived that can replicate autonomously due to eukaryotic ORIs (origin of replication).
• yeast vectors are called YRp vectors (Yeast replicating plasmids) or ARS vectors (autonomously replicating sequence).
• YEp vectors Yeast episonal plasmids
• the class of YAC vectors (Yeast Artifical Chromosome) behaves like independent chromosomes.
• a yeast vector containing a gene for expression so that it can be expressed is introduced into the yeast by transformation. Methods such as electroporation or the incubation of competent cells by vector DNA are suitable for this.
• Suitable yeast expression promoters are known to those skilled in the art. Examples include the SOD1 promoter (superoxide dismutase), ADH promoter (alcohol dehydrogenase), the promoter for the acid phosphatase gene, HXT2 promoter (glucose transporter 2), HXT7 promoter (glucose transporter 7), GAL2 promoter (galactose transporter) and other.
• the construct consisting of an expression promoter of a yeast and a gene for expression (e.g.
• GLUT4V85M is part of a yeast vector for the purpose of expression.
• this yeast vector can be present as a self-replicating particle independent of the genome of the yeast or can be stably integrated into the genome of the yeast.
• anyone is suitable as a yeast vector Polynucleotide sequence that can be propagated in a yeast.
• yeast plasmids or artificial yeast chromosomes can be used as yeast vectors.
• Yeast vectors generally contain an "origin of replication" (2 ⁇ , ars) for the initiation of replication and a selection marker which usually consists of an auxotrophy marker or an antibiotic resistance gene.
• pBM272, pCS19, pEMBCYe23, pFL26 are known to a person skilled in the art.
• the selection of a cell in the sense of this invention is to be understood as its targeted enrichment on the basis of a selection marker, for example resistance to an antibiotic or the ability to grow on a certain minimal medium, and furthermore its isolation and subsequent cultivation on an agar plate or in submerged culture.
• a selection marker for example resistance to an antibiotic or the ability to grow on a certain minimal medium, and furthermore its isolation and subsequent cultivation on an agar plate or in submerged culture.
• yeast Saccharomyces cerevisiae 17 hexose transporters and an additional three maltose transporters are known which, if they are expressed sufficiently enough, are able to transport hexoses into the yeast.
• a strain is known from which all transporters which are suitable for taking up hexose have been removed by deletion. This strain only contains the two genes MPH2 and MPH3, which are homologous to maltose transport proteins. The two genes MPH2 and MPH3 are repressed in the presence of glucose in the medium. Manufacture and Characterization of this yeast strain is in Wieczorke et all, FEBS Lett. 464, 123-128 (1999).
• This strain is unable to multiply on a substrate with glucose as the only source of carbon. From this strain, mutants can be selected which functionally express GLUT1 starting from a corresponding vector (strain hxt fgy1-1). If a plasmid vector which carries a GLUT4 gene under the control of a yeast promoter is transformed into the yeast strain hxt fgy1-1, very little glucose is nevertheless transported. The functional expression of GLUT4 requires further adjustments to this yeast strain in order to enable significant glucose transport using GLUT4. Such yeast strains, which take up glucose in cells by means of a single glucose transporter GLUT4, can be isolated on substrates with glucose as the only carbon source.
• a yeast strain hxt fgy1-1 which carries a GLUT4 gene under the functional control of a yeast promoter, is transformed.
• These yeast cells transformed in this way are placed on a nutrient medium which contains glucose as the sole carbon source and are incubated thereon. After a few days of incubation at, for example, 30 ° C., growth of individual colonies is observed. One of these colonies is isolated. If the yeast plasmid is removed from this colony, it does not multiply on the nutrient medium with glucose as the only source of carbon.
• this strain is in turn able to concentrate on a medium with glucose as the only carbon source multiply.
• yeast strains are the subject of international application PCT / EP02 / 01373 with the date of the application of February 9, 2002, which claims the priority of DE 10106718.6 of February 14, 2002.
• Yeast strains all of which own transporters for hexoses (glucose transporters) are no longer functional, are used in the German Collection of
• DSMZ Microorganisms and cell cultures GmbH
• polynucleotide and amino acid sequences for GLUT4 are accessible, for example, via the following entries in Genbank: M20747 (cDNA; human), EMBL: D28561 (cDNA; rat), EMBL: M23382 (cDNA; mouse), Swissprot: P14672 (protein; human), Swissprot: P19357 (protein; rat) and Swissprot: P14142 (protein; mouse).
• EMBL M20653 (cDNA; human), EMBL: M 13979 (cDNA; rat), EMBL: M23384 (cDNA; mouse), Swissprot: P11166 (protein ; Human), Swissprot: P11167 (protein; rat) and Swissprot: P17809 (protein; mouse).
• Medicines are dosage forms of pharmacologically active substances for the treatment of diseases or physical malfunctions in humans and animals.
• oral therapy for example, powders, granules, tablets, pills, lozenges, coated tablets, capsules, liquid extracts, tinctures, syrups are known.
• aerosols, sprays, gels, ointments or powders are used.
• Parenteral use is possible with injection or infusion solutions with ampoules, bottles or spray ampoules.
• These and other medicinal products are known to those skilled in pharmaceutical technology (Galenics).
• auxiliaries for the formulation of a drug enable the preparation of the active substance with the aim of enabling the active ingredient to be applied, distributed and developed in an optimal manner for the particular application.
• auxiliaries are, for example, fillers, binders, disintegrants, or lubricants such as lactose, sucrose, mannitol, sorbitol, cellulose, starch, dicalcium phosphate, polyglycols, alginates, polyvinylpyrrolidone, carboxymethyl cellulose, talc or silicon dioxide.
• Diabetes or diabetes is manifested by the excretion of glucose in the urine when there is an abnormal increase in blood glucose (hyperglycaemia) due to a chronic metabolic disorder due to a lack of insulin or a reduced insulin effect.
• the lack of or reduced insulin action leads to deficient absorption and utilization of the glucose absorbed into the blood by the body cells.
• lipolysis increases in the adipose tissue with an increase in the free fatty acids in the blood.
• Obesity is an abnormal weight gain due to a disturbed energy balance due to excessive calorie intake, which involves a health risk.
• the amount of a hexose which is taken up by a yeast strain provided as described above can be determined by means of intake studies with radioactively labeled glucose.
• a certain concentration of the yeast cells for example an amount of 60 mg wet weight per ml, is suspended in, for example, 100 ⁇ l of a buffer and a defined amount of ⁇ C- or 3H-labeled glucose is added as the only carbon source.
• the cells are incubated and defined amounts of the cells are removed at certain times.
• the amount of glucose consumed is determined using LSC (Liquid Scintillation Counting).
• the determination of the amount of a hexose which is taken up by a yeast strain provided as just described can also be carried out by means of a growth test on media with glucose as the sole carbon source.
• the growth rate of the strain after adding the compound is determined, for example, by regular measurements of the optical density of the culture at 600 nm and this value is compared to the growth rate of a control strain (e.g. wild-type yeast strain).
• a compound is provided in particular by chemical synthesis or isolation of chemical substances from biological organisms.
• the chemical Synthesis can also be automated.
• the compounds obtained by synthesis or isolation can be dissolved in a suitable solvent.
• suitable solvents are in particular aqueous solutions which contain a certain proportion of an organic solvent such as DMSO (dimethyl sulfoxide).
• Seq ID No. 1 discloses a polynucleotide sequence comprising the coding region of the protein GLUT4V85M.
• Seq ID No. 2 discloses the amino acid sequence of the protein GLUT4V85M.
• Seq ID No. 3 discloses the polynucleotide sequence of the vector p4H7GLUT4V85M.
• yeast strains described in the present work came from the strain CEN-PK2-1C (MATa leu2-3, 112 ura3-52 trp1-289 his3- ⁇ 1MAL2-8 c SUC2).
• the production of a yeast strain with deletions in the hexose transporter genes (HXT) was carried out by Wieczorke et al., FEBS Lett.
• the media was based on 1% yeast extract and 2% peptone (YP), while the minimal media consisted of 0.67% Difco yeast nitrogen-free, non-amino acid (YNB) and additives for Auxotrophy needs as well as different carbon sources included.
• the yeast cells were grown under aerobic conditions at 30 ° C on a rotary shaker or on agar plates. Cell growth was monitored by measuring the optical density at 600 nm (OD ⁇ D O ) or determining the diameter of the yeast colonies.
• Glucose transport was measured as the uptake of D- [U- 14 C] glucose (Amersham) and the kinetic parameters were determined from Eadie-Hofstee graphics.
• the cells were centrifuged, washed with phosphate buffer and resuspended in phosphate buffer at a concentration of 60 mg (wet weight) per ml.
• the glucose uptake was determined at glucose concentrations between 0.2 and 100 mM, and the specific activity of the substrate ranged between 0.1 and 55.5 kBq ⁇ mol "1.
• the cells and the glucose solutions were preincubated at 30 ° C.
• a new heterologous expression system for glucose transporters from mammalian cells has been developed. This system is based on a S. cerevisiae strain from which all endogenous glucose transporters have been removed by destroying the coding genes. This strain is no longer able to absorb glucose via the plasma membrane and to grow with glucose as the only carbon source.
• the heterologous glucose transporters of humans or others To integrate mammals, GLUT1 and GLUT4, in an active form into the plasma membrane of yeast, additional mutations had to be introduced into the yeast strain.
• GLUT1 is only active in a fgy1-1 mutant strain, GLUT4 only in fgy1-1 fgy4-X double mutants.
• the FGY1 gene could be cloned. It is the S. cerevisiae ORF YMR212c. The results show that either Fgy1 or a product produced by Fgy1 inhibits the activity of human glucose transporters or is involved in the fusion of the GLUT-transporting vesicles with the plasma membrane.
• GLUT4 proteins in yeast are located in intracellular structures.
• a total of nine recessive mutants could be isolated (fgy4-1 to fgy4-9), in which GLUT4 is now passed on to the plasma membrane and becomes active there with a simultaneous fgy1-1 mutation.
• This enzyme, the sterol-C-24 (28) - reductase catalyzes the last step of the ergosterol biosynthesis and converts Ergosta-5,7,22,24, (28) -tetraenol into the end product ergosterol.
• the Erg4 protein probably contains eight transmembrane domains and is located in the endoplasmic reticulum.
• An erg4 mutant is viable because the incorporation of the ergosterol precursors into the yeast membranes compensates for the loss of ergosterol.
• the inhibitory influence of Erg4 on the functionality of GLUT4 was confirmed by a targeted erg4 deletion in the hxt fgy1-1 strain.
• the resulting strain (hxt fgy1-1 ⁇ erg4) was designated SDY022.
• Table 3 shows the description of the yeast strains deposited in connection with this patent application at the German Collection of Microorganisms and Cell Cultures (DSMZ) - Mascheroder Weg 1 b 38124 Braunschweig. Table: Growth of GLUT1 and GLUT4 transformants on glucose medium
• Table 2 Growth of GLUT1 and GLUT4 transformants on glucose medium with and without ergosterol under anaerobic conditions
• Table 3 Characteristics of the deposited yeast strains (Saccharomyces cerevisiae)
• Base medium 0.67% Yeast Nitrogen Base without amino acids (Difco); pH 6.2. Supplementation of auxotrophies: leucine (0.44 mM), tryptophan (0.19 mM), histidine (0.25 mM9, uracil (0.44 mM). Maltose can be used between 1-2%.
• This international depository accepts the microorganism designated under 1, which it received on 2002-09-03 (date of first deposit) 1 .
• microorganism referred to under I was received by this International Depository on (date of first filing) and an application for the conversion of this first filing into a deposit under the Budapest Treaty was received on (date of receipt of the request for conversion).
• microorganism referred to under I was received by this international depository on (date of first deposit) and an application for conversion of this first deposit into a deposit according to the Budapest Treaty was received on (date of receipt of the application for conversion).
• rule S.4 letter d is the time at which the status of an international depository was acquired.
• microorganism referred to under 1 was received by this international depository on (date of first filing) and an application for the conversion of this first filing into a deposit according to the Budapest Treaty was received on (date of receipt of the application for conversion).
• microorganism referred to under I was received by this international depository on (date of first deposit) and an application for conversion of this first deposit into a deposit according to the Budapest Treaty was received on (therefore the receipt of the request for conversion).
• microorganism referred to under I was received by this international depository on (date of first filing) and an application for conversion of this first filing into a deposit under the Budapest Treaty was received on (date of receipt of the application for conversion).

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