CN1694960A - Use of saccharomyces cerevisiae ERG4 mutants for expressing mammalian glucose transporters - Google Patents

Abstract

The invention relates to yeast strains in which a human GLUT4 transporter or a human GLUT1 transporter can be functionally expressed and in particular GLUT4 transport proteins which can be particularly easily functionally expressed in yeast strains.

Description

The purposes of yeast saccharomyces cerevisiae ERG4 mutant in the glucose transporter of expressing the Mammals source

Invention field

The present invention relates to can functional expression people GLut4 and the yeast strain of GLut1 translocator.

Background technology

Most heterotrophic cells via specific translocator with glucose transport in cell.Different Biological Development goes out the mechanism of different mediation glucose transports, as the collaborative haulage system of proton particularly, Na ⁺The system of glucose transporter, conjugated protein dependence, phosphotransferase system and be used for the system of facilitation diffusion.In eukaryote, mediate the glucose uptake that spreads via facilitation by GLUT gene in the mammalian body (GLUT=glucose transporter) coding with by the glucose transporter family that HXT gene (HXT=hexose translocator) in the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is encoded.Described translocator belongs to bigger HUCEP-8 family.They are characterised in that and have 12 transbilayer helixs and a plurality of conservative amino acid residues.Glucose transport is played the part of important role in the disorder relevant with glucose stable state defective (as diabetes or Fanconi-Bickel syndromes).Therefore, the intravital glucose transport of Mammals has become the theme of many researchs.Up to the present, 13 kinds of glucose transporter sample protein (GLUT1 is to GLUT12, HMIT-H-inositol translocator) have been identified.Described translocator is being played the part of crucial role in the following areas, comprises glucose uptake is advanced various tissues, glucose storage, myocyte and the adipocyte in liver to the measurement to glucose of the insulin-dependent picked-up of glucose and pancreas beta cell.

The GLUT1 mediation is advanced glucose transport red corpuscle and is passed through hemato encephalic barrier, but GLUT1 also is expressed in many other tissues, and GLUT4 is limited to insulin-dependent sex organization, mainly is muscle and fatty tissue.In described insulin-dependent sex organization, the intracellular region chamber of control GLUT4 translocator or plasma membrane compartment orientation are a kind of mechanism of important adjusting glucose uptake.In the presence of Regular Insulin, GLUT4 heavily distributes and passes plasma membrane in the born of the same parents, so that promote glucose uptake.GLUT1 is expressed in described insulin-dependent sex organization equally, and it influences by Regular Insulin equally in intracellular distribution, though influence intensity and not as GLUT4.In addition, the relative efficiency of GLUT1 or GLUT4 catalysis sugar transport is not only determined by the degree of each translocator guiding cell surface, determined by their dynamics.

Different glucose transporter isotypes has been made the effect of various glucose transporter isotypes in the relevant insulin-dependent sex organization and the research of definite character become complicated by the true and glucose metabolism rapidly of coexpression.In order to solve these difficult problems, used heterologous expression system as xenopus (Xenopus) ovocyte, tissue culture cells, insect cell and yeast cell and so on.But some have occurred and it seems the difficulty relevant with these systems: the glucose transporter of too low, the described system self of the activity of the translocator of heterogenous expression, the translocator of significant proportion are trapped in the born of the same parents or even produce the translocator of non-activity.

Can in Wine brewing yeast strain, express the GLUT4 protein of natural Mammals GLUT4 protein, particularly people under given conditions with functional mode.

Yeast cell is a unicellular eukaryote.Therefore, they are more suitable for expressing some protein than bacterial system, when particularly relating to the shaker test of carrying out the identification of pharmaceutical active material.

Summary of the invention

The present invention relates to purifying and isolating polynucleotide, it contains coding GLUT4V85M protein DNA sequence.

Described protein contains in 85 positions of people GLUT4 gal4 amino acid chain by the amino acid exchange of Xie Ansuan to methionine(Met).The GLUT4V85M protein of this change provides other possibility for expression has the GLUT4 protein of function.If the Wine brewing yeast strain of self glucose transporter complete deactivation (=hxt (-)) can be observed the picked-up that has glucose after expressing described GLUT4 protein, should think that then GLUT4 protein has function with regard to yeast saccharomyces cerevisiae.Can be by the transhipment of radio-labeling glucose being measured or by measuring glucose uptake as the growth on the substratum of sole carbon source with glucose.

In a preferred embodiment, purifying and the isolating polynucleotide that comprise the dna sequence dna of coding GLUT4V85M protein can include or contain following sequence:

A) according to the nucleotide sequence of Seq ID No.1,

B) under the stringency condition with the sequence hybridization of Seq ID No.1 and the proteinic nucleotide sequence of the GLUT4V85M that encodes.

Purifying and isolating polynucleotide optimized encoding have the GLUT4V85M protein of the aminoacid sequence of Seq ID No.2.

Purifying can be connected (operationally link) with the promotor operability with the isolating polynucleotide that contain the GLUT4V85M protein DNA sequence of encoding as previously discussed.Suitable promotor is protokaryon or eukaryotic promoter especially, as Lac-, trp-, ADH-or HXT7 promotor.If bacterium or eukaryote can be translated into the proteinic mRNA of GLUT4V85M by described promotor generation under the assistance of carrier, the proteinic polynucleotide part of the GLUT4V85M that then encodes accurately is connected with the promotor operability.The example of this carrier is p4H7GLUT4V85M (Seq ID No.3) carrier.Can in yeast cell, express GLUT4V85M protein by described carrier.

In a preferred embodiment, the polynucleotide that contain the dna sequence dna of coding GLUT4V85M protein described above are suitable for duplicating in yeast cell, or are suitable for expressing the proteinic polynucleotide part of coding GLUT4V85M so that GLUT4V85M to be provided protein in yeast cell.Yeast cell from yeast saccharomyces cerevisiae is suitable especially.In order to duplicate in yeast cell and to express, the polynucleotide that contain the dna sequence dna of coding GLUT4V85M protein exist with the form of yeast vector.Coding GLUT4V85M proteinic polynucleotide region can be connected described promotor such as ADH promotor (alcoholdehydrogenase promotor) or HXT7 promotor (hexose translocator promotor) with the special promotor operability of yeast cell.Yeast vector is that exploitation is used for one group of carrier at the yeast cloned DNA.

The invention still further relates to the yeast cell from yeast saccharomyces cerevisiae, wherein all glucose transporter no longer have function (=hxt (-)) and do not contain functional Erg4 protein.Preferred this yeast cell is to be preserved in DSMZ (Germany microbial preservation center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH as yeast saccharomyces cerevisiae DSM 15187, Mascheroder Weg 1b, 38124 Brunswick, Germany)) yeast cell.

The invention still further relates to its all glucose transporter no longer has function and does not contain functional Fgy1 and the proteinic yeast cell of functional Erg4.The proteinic shortage of Erg4 protein or Fgy1 especially can be by the partially or completely removal in the interruption in corresponding genome encoding zone or described genome encoding zone and is caused.

For not containing functional glucose transporter, functional Fgy1 protein and the proteinic yeast cell of functional Erg4, the preferred yeast cell that is preserved in DSMZ with yeast saccharomyces cerevisiae DSM 15184 that uses.

Aforesaid yeast cell is preferred for expressing Mammals GLUT1 protein or Mammals GLUT4 protein, particularly from the protein of rat, mouse, rabbit, pig, ox or primates.An embodiment preferred is used yeast cell to express human GLUT4 or GLUT1 protein.

The brewing yeast cell that its whole glucose transporter and Erg4 protein no longer have function can contain polynucleotide of the present invention, and wherein these polynucleotide contain coding GLUT4V85M protein DNA sequence.Described yeast cell also can be expressed GLUT4V85M protein and therefore be contained this protein.

Yeast saccharomyces cerevisiae DSM 15185 yeast strains that preferably are preserved in DSMZ are as this yeast strain that contains the polynucleotide of the dna sequence dna that comprises coding GLUT4V85M protein.

Can prepare the yeast cell that its whole glucose transporter and Erg4 protein no longer have function and contains the polynucleotide of the dna sequence dna that comprises coding GLUT4V85M protein by following method:

A) yeast cell that provides its whole glucose transporter and Erg4 protein no longer to have function,

B) provide separation and the purified polynucleotides that can duplicate in yeast cell, these polynucleotide contain coding GLUT4V85M protein DNA sequence,

C) use from b) polynucleotide be converted from a) yeast cell,

D) select transformed yeast cells,

E) express GLUT4V85M protein suitably the time.

The carrier that the polynucleotide of the dna sequence dna that contains coding GLUT4V85M protein of separation and purifying preferably can duplicate in yeast cell, wherein said dna sequence dna is cloned in this carrier.The example of this carrier is p4H7GLUT4V85M (Seq ID No.3).

The invention still further relates to the yeast cell that its whole glucose transporter and Fgy1 and Erg4 protein no longer have function, wherein this yeast cell contains the polynucleotide of the dna sequence dna that comprises coding GLUT4V85M protein.Described yeast cell also can be expressed GLUT4V85M protein and therefore be contained this protein.This yeast strain is preferably the yeast saccharomyces cerevisiae DSM15186 that is preserved in DSMZ.

Can prepare the yeast cell that its whole glucose transporter and Fgy1 and Erg4 protein no longer have function and contain the polynucleotide of the dna sequence dna that comprises coding GLUT4V85M protein by following method:

A) yeast cell that provides its whole glucose transporter and Fgy1 and Erg4 protein no longer to have function,

B) provide separation and the purified polynucleotides that can duplicate in yeast cell, these polynucleotide contain coding GLUT4V85M protein DNA sequence,

C) use from b) polynucleotide be converted from a) yeast cell,

D) select transformed yeast cells,

E) express GLUT4V85M protein suitably the time.

The carrier that the polynucleotide of the dna sequence dna that contains coding GLUT4V85M protein of above-mentioned separation and purifying preferably can duplicate in yeast cell, wherein said dna sequence dna is cloned in this carrier.The example of this carrier is p4H7GLUT4V85M (Seq ID No.3).

The invention still further relates to the yeast cell that its whole glucose transporter no longer have function, wherein said yeast cell contains the polynucleotide of the dna sequence dna that comprises coding GLUT4V85M protein.

Described yeast cell also can be expressed GLUT4V85M protein and therefore be contained this protein.Preferred this yeast strain is yeast saccharomyces cerevisiae DSM 15188 bacterial strains that are preserved in DSMZ.

Can prepare the yeast cell that its whole glucose transporter no longer have function and contain the polynucleotide of the dna sequence dna that comprises coding GLUT4V85M protein by following method:

A) provide its yeast cell that whole glucose transporter no longer have function,

B) provide separation and the purified polynucleotides that can duplicate in yeast cell, these polynucleotide contain coding GLUT4V85M protein DNA sequence,

C) use from b) polynucleotide be converted from a) yeast cell,

D) select transformed yeast cells,

E) express GLUT4V85M protein suitably the time.

The carrier that the polynucleotide of the dna sequence dna that contains coding GLUT4V85M protein of separation and purifying preferably can duplicate in yeast cell, wherein said dna sequence dna is cloned in this carrier.The example of this carrier is p4H7GLUT4V85M (Seq ID No.3).

The invention still further relates to the protein that has according to the aminoacid sequence of Seq ID No.2.This protein is people GLUT4 protein, and wherein the Xie Ansuan in amino acid chain 85 positions is substituted by methionine(Met).

The invention still further relates to the method for identifying the compound that stimulates the GLUT4 protein active, it comprises step:

A) provide its whole glucose transporter and Erg4 protein no longer to have function and contain the yeast cell of the polynucleotide of the dna sequence dna that comprises coding GLUT4V85M protein,

B) provide chemical compound,

C) with a) yeast cell and b) chemical compound contact,

D) zymic glucose uptake mensuration c),

E) with d) the glucose uptake detected value with not with b) described in chemical compound contacted a) described in the glucose uptake detected value of yeast cell compare, cause d) described in the compound that increases of the glucose uptake amount of yeast cell stimulate described GLUT4 activity of proteins.Can expect stimulating the compound of GLUT4V85M protein active also to stimulate the GLUT4 activity.

The present invention also relates to contain with the aforesaid method compounds identified and be used for the additive of compounding pharmaceutical and the medicine of vehicle.In addition, the present invention relates to be used for the treatment of purposes in the medicine of I type and/or type ii diabetes in production with the aforesaid method compounds identified.

The present invention also relates to contain the aforesaid method compounds identified and be used for the additive of compounding pharmaceutical and the medicine of vehicle.In addition, the present invention relates to the aforesaid method compounds identified and be used for the treatment of purposes in the medicine of diabetes in production.

The present invention relates to the aforesaid method compounds identified in addition and is used for the treatment of purposes in the medicine of diabetes in production.The present invention also comprises the method for identifying the proteinic compound that suppresses the Erg4 genes encoding, and it comprises step:

A) provide its yeast cell that whole glucose transporter no longer have function, wherein this yeast cell contains dna sequence dna that comprises coding GLUT4V85M protein and the polynucleotide that can duplicate in yeast cell,

B) provide chemical compound,

C) with a) yeast and b) chemical compound contact,

D) zymic glucose uptake mensuration c),

E) with d) the glucose uptake detected value and not with b) described in chemical compound contacted a) described in yeast cell the glucose uptake detected value relatively, cause d) described in the compound that increases of zymic glucose uptake amount suppress the Erg4 activity of proteins.

The present invention relates to the method for the compound of identifying the respective egg white matter that suppresses the Fgy1 gene in addition, and it comprises step:

A) provide its whole glucose transporter and Erg4 protein no longer to have function and contain the proteinic yeast cell of GLUT4,

B) provide chemical compound,

C) with a) yeast and b) chemical compound contact,

D) zymic glucose uptake mensuration c),

E) with d) the glucose uptake detected value and not with b) described in chemical compound contacted a) described in yeast cell the glucose uptake detected value relatively, cause d) described in the compound that increases of zymic glucose uptake amount suppress the Fgy1 activity of proteins.

The invention still further relates to and contain the aforesaid method compounds identified and be used for the additive of compounding pharmaceutical and the medicine of vehicle.

Below with regard to the present invention of ins and outs more detailed description.

Hybridization is meant that two nucleic acid strands with complementary base sequence are assembled into two strands.Hybridization can be carried out between two DNA chains, DNA chain and a RNA chain and two RNA chains.In principle, may be the related nucleic acid of double chain form at first by heating, can prepare hybrid molecule, for example in water-bath, boiled 10 minutes, resolve into single chain molecule up to them with secondary structure.Subsequently, can slowly cool off them.At cooling stages, the complementary strand pairing forms double-stranded hybrid molecule.Under laboratory condition, usually under the assistance of hybridization filter membrane, hybridize, wherein by trace or electrophoresis with the polynucleotide application of sample of strand or sex change on the hybridization filter membrane.Can use suitable complementary polynucleotide molecule, this polynucleotide molecule by will be to be hybridized adds that the radioactivity fluorescent mark shows hybridization.Stringency is described the degree of mating or aliging under the specified conditions.High stringency has higher requirement than low stringency on coupling.According to using and purpose, set the specified conditions that are used for nucleic acid hybridization with different stringencies.Under high stringency, the hybridization condition of setting makes to have only mates to such an extent that extraordinary complementary molecule can be hybridized each other.Low stringency also can partly be hybridized the molecule with big relatively non-matching or wrong pairing base portion.

Particularly, if 68 ℃ of hybridization is at least two hours in containing the aqueous solution of 2xSSC, under room temperature, carry out among the 2xSSC/0.1%SDS subsequently for the time 5 minutes first time washing, washed 1 hour in 1xSSC/0.1%SDS at 68 ℃ then, washed in 0.2%SSC/0.1%SDS 1 hour at 68 ℃ then, then this hybridization conditions is considered to tight again.

Prepare 2xSSC, 1xSSC or 0.2xSSC solution by suitable dilution 20xSSC solution.20xSSC solution contains 3mol/l NaCl and 0.3mol/l Trisodium Citrate.PH is 7.0.The technician is familiar with the method at stringency condition hybridization polynucleotide.Suitable guidance is found in professional handbook, particularly as " Current Protocols in Molecular Biology " (Wiley Interscience; Editor: Frederich M.Ausubel, Roger Brant, Robert E.Kingston, David J.Moore, J.G.Seidmann, Kevin Struhl; ISBN:0-471-50338-X).

Yeast vector can be divided into different subgroups.YIp carrier (yeast integrative plasmid) still contains selective yeast genes (as URA3, LEU2) basically corresponding to the carrier that is used for cloning on bacterium.

After introducing described carrier, to have only when foreign DNA to be integrated in the yeast chromosomal, these sequences are just together duplicated with karyomit(e), and along with clone's formation stably passes to all daughter cells.

Based on this method, derive because eucaryon ORI (replication orgin) and plasmid that can self-replicating.This yeast vector is called as YRp carrier (yeast replicating plasmid) or ARS carrier (autonomously replicating sequence).In addition, also have derived from yeast 2 μ m plasmids and contain selected marker's YEp carrier (yeast additive type plasmid).The behavior of YAC (yeast artificial chromosome) class carrier is as karyomit(e) independently.

To contain the yeast vector of wanting expressing gene by the mode that transforms and introduce yeast, so that can expressing said gene.The example that is fit to the method for this purpose is electroporation or hatches competent cell with carrier DNA.The technician knows suitable yeast expression promotor, and example is the promotor, HXT2 (glucose transporter 2) promotor, HXT7 (glucose transporter 7) promotor, GAL2 (semi-lactosi translocator) promotor of SOD1 (superoxide-dismutase) promotor, ADH (alcoholdehydrogenase) promotor, acid phosphatase gene and other.In order to express purpose, the construct that contains yeast expression promotor and the gene (as GLUT4V85M) that will express is the part of yeast vector.In order to realize expressing, described yeast vector can be to be independent of the self replication particulate of yeast genes group or can to advance the yeast genes group by stable integration.In principle, suitable yeast vector is any polynucleotide sequence that can breed in yeast.Operable yeast vector is yeast plasmid or yeast artificial chromosome especially.Yeast vector contain usually replication orgin (2 μ, ars) or the starting point of initial reproduction process and comprise the selected marker of auxotrophy mark or antibiotics resistance gene usually.The example of the yeast vector that the technician knows is pBM272, pCS19, pEMBCYe23, pFL26, pG6, pNN414, pTV3, p426MET25, p4H7 and other.

According to the present invention, the selection of cell be meant by selected marker (as antibiotics resistance or grow in ability on the special minimum medium) with further separates and subsequently cultivation (on agar plate, it is cultivated or deep layer is cultivated) and to it carry out specific concentrated.

The selection of cultivation, conversion and transformed yeast cells also has the expression of protein in yeast cell to belong to the conventional method of using of technician.Be found in the textbook of standard about the explanation of described method, as Walker Graeme M.: " Yeast Physiology and Biotechnology " (Wileyand Sons, ISBN:0-471-9446-8) or " Protein Synthesis and Targeting inYeast " (Alistair J.P.Brown, Mick F.Fruite and John E.G.Mc Cartly compile; Springer Berlin; ISBN:3-540-56521-3) or " " Methods in Yeast Genetics ", 1997:A Cold Spring Harbor Laboratory Course Manual; Adams Alison (editor); Cold Spring Harbor Laboratory; ISBN:0-87969-508-0 ".

Yeast saccharomyces cerevisiae has 17 kinds of known hexose translocators and 3 kinds of known maltose translocators, is expressing under the sufficiently high situation, and described maltose translocator can be transported hexose into described yeast.In a kind of known bacterial strain, removed all by disappearance and be suitable for the translocator of hexose picked-up.This bacterial strain only contains two kinds with the gene M PH2 and the MPH3 that come from maltose translocator matter.When having glucose in the substratum, MPH2 and MPH3 two genes are suppressed.People such as Wieczorke (FEBS Lett.464,123-128 (1999)) have described preparation and the feature of this yeast strain.This bacterial strain can not be bred containing on the substratum of glucose as sole carbon source.Can originate in this bacterial strain mutant (hxt fgy1-1 bacterial strain) of respective carrier selection function expression GLUT1.If use the plasmid vector that carries the GLUT4 gene under the Yeast promoter control to transform hxt fgy1-1 yeast strain, still have only very small amount of glucose to be transported.For this yeast strain, need further to adjust functional GLUT4 and express, so that realization is by the significant glucose transport of GLUT4 means.Can separate the yeast strain of this its cell on as the substratum of sole carbon source having glucose by single GLUT4 glucose transporter ingestion of glucose.For this purpose, with the GLUT4 gene transformation hxt fgy1-1 bacterial strain that is under the functional control of Yeast promoter.With these in this way transformed yeast cells be added on and contain on the nutritional medium of glucose as sole carbon source, and hatch thereon.For example 30 ℃ hatch several days after, observe the growth of single bacterium colony.Separate in these bacterium colonies.From described bacterium colony, remove the yeast plasmid prevention and containing glucose as the breeding on the nutritional medium of sole carbon source.If transform the bacterial strain that this no longer contains vector plasmid once more with carrying the yeast vector that is in the GLUT4 gene under the functional control of Yeast promoter, so described bacterial strain can be once more bred containing on the substratum of glucose as sole carbon source.

Above-mentioned yeast strain is the theme that is filed in the International Application PCT/EP02/01373 on February 9th, 2002, and this application requires the right of priority of the DE 10106718.6 on February 14th, 2002.

The yeast strain that self whole hexose translocator (glucose transporter) no longer has a function relevantly with International Application PCT/EP02/01373 is preserved in Germany microbial preservation center (DSMZ) with preserving number DSM14035, DSM 14036 or DSM 14037 in more early date.

The polynucleotide of GLUT4 and aminoacid sequence can for example be obtained via following clauses and subclauses: GeneBank:M20747 (cDNA; The people), EMBL:D28561 (cDNA; Rat), EMBL:M23382 (cDNA; Mouse), Swissprot:P14672 (protein; The people), Swissprot:P19357 (protein; Rat) and Swissprot:P14142 (protein; Mouse).

The polynucleotide sequence of GLUT1 and aminoacid sequence are disclosed under database code shown below: EMBL:M20653 (cDNA; The people), EMBL:M13979 (cDNA; Rat), EMBL:M23384 (cDNA; Mouse), Swissprot:P11166 (protein; The people), Swissprot:P11167 (protein; Rat) and Swissprot:P17809 (protein; Mouse).

Medicine is the agent shape of the pharmacological active substance of treatment humans and animals disease or physical function obstacle.The example of the dosage form of oral administration is powder, granule, tablet, pill, lozenge, sugar coated tablet, capsule, fluid extract, tincture and syrup.The example that is used for external application is aerosol, sprays, gelifying agent, ointment or powder.Use bottle, bottle or prefilled syringe, offer medicine outward but the solution of injectable or infusion can carry out gi tract.For the technician of technical field of pharmaceuticals, these and other medicine is known.

The vehicle that is used for compounding pharmaceutical makes and can prepare active substance so that using, distributing and acting on for application-specific optimization activeconstituents.The example of this class vehicle is weighting agent, tackiness agent, disintegrating agent or glidant, as lactose, sucrose, N.F,USP MANNITOL, Sorbitol Powder, Mierocrystalline cellulose, starch, secondary calcium phosphate, polyoxyethylene glycol, alginate, polyvinylpyrrolidone, carboxy methyl cellulose, talcum powder or silicon-dioxide.

Owing to be that insulin deficit or insulin action reduce the chronic metabolic trouble that causes, diabetes show as glucose and increase (hyperglycemia) unusually with urine discharge and blood glucose levels.The shortage of insulin action or reduction cause cell picked-up to be advanced the absorption and the conversion deficiency of the glucose of blood.In fatty tissue, the insulin resistance hormone has the effect that increases lipolysis, follows the increase of free fatty acid levels in the blood.

Greasiness (obesity) is that the unbalance body weight that causes of energy that the picked-up heat too much causes increases unusually, and obesity constitutes a threat to health.

Can use the hexose amount of radiolabeled glucose assays by the means of picked-up research by the yeast strain that is provided (as just describing) picked-up at preamble.For this purpose, the yeast cell of specific concentrations is suspended in as the concentration of every milliliter of 60mg weight in wet base (for example with) in the 100 μ l damping fluids, and is mixed into really quantitative ¹⁴C-or ³The glucose of H-mark is as unique carbon source.Incubated cell, and take out true quantitative cell at specified time.Under the help of LSC (liquid scintillation counter), measure the glucose amount of picked-up.Yet, also can by contain glucose as the growth experiment on the substratum of sole carbon source measure by provided and as the hexose amount of the yeast strain picked-up just described at preamble.For this purpose, after adding compound, measure the growth velocity (for example measuring the optical density(OD) of culture at regular intervals at 600nm) of bacterial strain, and this value is compared with the growth velocity of control strain (as the yeast wild type strain).

Compound is provided, particularly synthetic or by separation chemistry material from biological organism by chemical mode.Also can carry out chemosynthesis in automatic mode.Can will pass through the compound dissolution of synthetic or separation acquisition in suitable solvent.Suitable solvent especially contains the aqueous solution of the organic solvent (as DMSO (dimethyl sulfoxide (DMSO))) of specified proportion.

For with the compound treatment yeast strain so that identify according to for the compound of aforementioned invention, can in the automatic laboratory system that for this reason provides, carry out especially.This system can comprise the cell with pit or microtiter plate, Eppendorf pipe or the laboratory glassware of specific preparation.Automatically laboratory system is generally the design of high-throughput speed.Therefore, the method for under automatic laboratory system is assisted, carrying out, for example aforesaid method is also referred to as HTS (high throughput screening).

Seq ID No.1 discloses the polynucleotide sequence that contains the GLUT4V85M protein coding region.Seq ID No.2 discloses the proteinic aminoacid sequence of GLUT4V85M.Seq ID No.3 discloses the polynucleotide sequence of p4H7GLUT4V85M carrier.

Embodiment

The use of yeast strain

All yeast strains described herein are all from bacterial strain CEN-PK2-1C (MATa leu2-3,112 ura3-52 trp1-289 his3-Δ 1MAL2-8 ^CSUC2).The preparation of the yeast strain of hexose translocator (HXT) genetically deficient is described (FEBS Lett.464 by people such as Wieczorke, 123-128 (1999): EBY-18ga (MATa Δ hxt1-17 Δ gal2 Δ agt1 Δ stl1 leu2-3,112 ura3-52trp1-289 his3-Δs, 1 MAL2-8 ^CSUC2), EBY.VW4000 (MATa Δ hxt1-17 Δ gal2 Δ agt1 Δ mph2 Δ mph3 Δ stl1 leu2-3,112 ura3-52 trp1-289 his3-Δs, 1 MAL2-8 ^CSUC2)).Substratum is based on 1% yeast extract and 2% peptone (YP), and minimum medium is made up of no amino acid whose 0.67% Difco yeast nitrogen (YNB), and contains required additive of auxotrophy and different carbon sources.Yeast cell grows on gyrate shaker or the agar plate under 30 ℃, aerobic conditions.By at 600nm measuring light density (OD ₆₀₀) or the diameter of measuring yeast colony monitor the cell growth.

The mensuration of glucose uptake

With to D-[U- ¹⁴C]-picked-up of glucose (Amersham) measures glucose transport, and measures kinetic parameter from Eadie-Hofstee figure.The centrifugal cell of removing washs with phosphoric acid buffer, and is resuspended to phosphoric acid buffer with the concentration of every milliliter of 60mg weight in wet base.The picked-up of glucose concentration determination glucose at 0.2 to 100mM, and the ratio vigor of substrate is between 0.1 to 55.5kBq μ mol ^-130 ℃ of pair cells and glucose solution preincubate 5 minutes.By adding radioactivity glucose and initial glucose uptake to cell.Add ice-cooled stop buffer (the 0.1M KiPO of 10ml after hatching for 5 seconds ₄, pH6.5,500mM glucose) and by (Φ=24mm Whatman) goes up filtration and rapid emigrated cells at glass fibre filter.With the rapid washing nozzle of ice-cooled damping fluid 3 times, the radioactivity of using liquid scintillation counter measurement to mix.Cell in the presence of the inhibitor or only having hatch 15 minutes in the presence of the solvent after, use 50mM or 100mM radioactivity glucose in 15 seconds picked-up test, to measure the inhibition of cytochalasin B (final concentration 20 μ M are dissolved in the ethanol).

Developed the new system of the glucose transporter that heterogenous expression mammalian cell source.This system in described bacterial strain, by destroying encoding gene, has removed all endogenous glucose transporter based on Wine brewing yeast strain.This bacterial strain no longer can and no longer can be grown as sole carbon source with glucose via the plasma membrane ingestion of glucose.For people or other mammiferous allos glucose transporter GLUT1 and GLUT4 are integrated into the yeast plasma membrane with activity form, must introduce extra sudden change to yeast strain.GLUT1 only just has activity in the fgy1-1 mutant strain, GLUT4 only just has activity in the two mutant strains of fgy1-1 fgy4-x.

Cloned the FGY1 gene.It is yeast saccharomyces cerevisiae ORF YMR212c.With regard to function, the result shows the activity that the product inhibition human glucose transport protein of Fgy1 or Fgy1 generation is white or relates to the fusion of GLUT transitional vesicle and plasma membrane.

Opposite with GLUT1 and similar in appearance to mammalian cell, the GLUT4 protein positioning of vast scale is in the cell inner structure in the yeast.Separated 9 kinds of cryptic mutants (fgy4-1 is to fgy4-9) altogether, wherein GLUT4 further is directed to plasma membrane this moment, and under the situation of following the fgy1-1 sudden change, has activity there.

Insertion gene pool (the Genes Dev.1994 that people such as Bruns describe; 8:1087-105) be used to complementation analysis.Use earlier the GLUT4 plasmid, transform hxt fgy1-1 bacterial strain with the insertion gene pool that moves then.Be the transformant that screening can be grown on dextrose culture-medium after this.As a result, the ERG4 gene is destroyed in the mutant of a research.The ERG4 biosynthetic enzyme of ergosterol (oxydo-reductase) of encoding.This enzyme (sterol-C-24 (28)-reductase enzyme) biosynthetic final step of catalysis ergosterol, with ergot steroid-5,7,22,24, (28)-tetraene alcohol changes the end product ergosterol into.Erg4 protein contains 8 membrane-spanning domains at present and is positioned endoplasmic reticulum.Because the ergosterol precursor mixes the forfeiture that the yeast plasma membrane can remedy ergosterol, so the erg4 mutant can be survived.

Proved conclusively the inhibition influence of Erg4 by the disappearance of specificity in hxt fgy1-1 bacterial strain erg4 to the GLUT4 function.The bacterial strain (hxt fgy1-1 Δ erg4) that produces is called as SDY022.

Protein interaction test under the Split-Ubiquitin system assists shows the direct and yeast Erg4 interaction of people GLUT4.Therefore, can infer the yeast Erg4 protein in the endoplasmic reticulum or directly stop the further displacement of GLUT4, or being that certain important mode is modified GLUT4 for displacement and/or function.

Confirm equally also that in Δ hxt bacterial strain only ERG4 disappearance (that is, although FGY1 has function) also can activate GLUT1, does not activate GLUT4.Growth test the results are summarized in table 1.

In order to get rid of ergosterol self GLUT4 is imposed negative impact, on the agar plate that is containing ergosterol under the anaerobic condition, carry out growth test.Under these conditions, any yeast strain that transforms with GLUT4 all can not grow (table 2).Opposite with grow aerobically, under anaerobic conditions the GLUT1 transformant in the hxtfgy1-1 bacterial strain does not show in glucose and grows.Only behind disappearance ERG4, the GLUT1 transformant can be grown.

Val85 that is undertaken by vitro mutagenesis and the exchange of Met make GLUT4 be independent of the fgy1-1 sudden change, even and produced also have a GLUT4V85M of function in hxt erg4 bacterial strain.This observation shows that Fgy1 directly or indirectly acts on this position, and this position is positioned in second transbilayer helix of GLUT translocator.

Table 3 is showed relevant be preserved in Germany microbial preservation center (DSMZ)-Mascheroder Weg 1b, 38124 Brunswick, the explanation of the yeast strain of Germany with present patent application.

Table 1:GLUT1 and the growth of GLUT4 transformant on dextrose culture-medium.

Genotype	1% glucose		1% glucose
					Δhxt?fgy1-1	????GLUT4	??-	????GLUT1	++
Δhxt?fgy1-1Δerg4	????GLUT4	??++	????GLUT1	++
					Δhxt?fgy1-1Δerg4	Carrier	??-	Carrier	-
Δhxt?fgy1-1Δerg5	????GLUT4	??-	????GLUT1	++
					Δhxt?fgy1-1Δerg4Δerg5	????GLUT4	??+	????GLUT1	++
Δhxt?Δerg4	????GLUT4	??-	????GLUT1	+
					Δhxt?Δerg5	????GLUT4	??-	????GLUT1	-

Table 2:GLUT1 and the growth of GLUT4 transformant on the dextrose culture-medium that has or do not have ergosterol under the anaerobic conditions.

Genotype	1% glucose	1% glucose+33mg/l ergosterol
			Δhxt?fgy1-1??????????????GLUT1 ??????????????????????????GLUT4	???- ???-	????- ????-
Δhxt?fgy1-1Δerg4????????GLUT1 ??????????????????????????GLUT4	???- ???-	????++ ????-
			Δhxt?fgy1-1Δerg5????????GLUT1 ??????????????????????????GLUT4	???- ???-	????- ????-
Δhxt?fgy1-1Δerg4Δerg5??GLUT1 ??????????????????????????GLUT4	???- ???-	????++ ????-
			ΔhxtΔerg4???????????????GLUT1 ??????????????????????????GLUT4	???- ???-	????(+) ????-
ΔhxtΔerg5???????????????GLUT1 ??????????????????????????GLUT4	???- ???-	????- ????-

Table 3: the feature of the yeast strain of preservation (yeast saccharomyces cerevisiae)

Be preserved in the preserving number of DSM Z	Genotype	Phenotype	Plasmid
				DSM 15187	?MATa?Δhxt1-17?Δgal2 ?Δagt1?Δstl1?Δmph2 ?Δmph3?Δerg4?leu2-3， ?112?ura3-52?trp1-289 ?his3-Δ1?MAL2-8 C?SUC2	Use 1% maltose as carbon source, strain growth; Be glucose, leucine, tryptophane, Histidine and uracil auxotrophy
DSM 15184	?MATa?Δhxt1-17?Δgal2 ?Δagt1?Δstl1?Δerg4 ?fgy1-1?leu2-3，112 ?ura3-52?trp1-289 ?his3-Δ1?MAL2-8 C?SUC2	Use 1% maltose as carbon source, strain growth; Be glucose, leucine, tryptophane, Histidine and uracil auxotrophy
				DSM 15185	?MATa?Δhxt1-17?Δgal2 ?Δagt1?Δstl1?Δmph2 ?Δmph3?Δerg4?leu2-3， ?112?ura3-52?trp1-289 ?his3-Δ1?MAL2-8 C?SUC2	Use 1% maltose as carbon source, strain growth; Be glucose, leucine, tryptophane and histidine auxotroph	P4H7GLUT4V 85M (selected marker URA3) ,=Seq ID No.3
DSM 15186	?MATa?Δhxt1-17?Δgal2 ?Δagt1?Δstl1?Δerg4 ?fgy1-1?leu2-3，112 ?ura3-52?trp1-289 ?his3-Δ1?MAL2-8 C?SUC2	Use 1% maltose as carbon source, strain growth; Be glucose, leucine, tryptophane and histidine auxotroph	P4H7GLUT4V 85M (selected marker URA3) ,=Seq ID No.3
				DSM 15188	?MATa?Δhxt1-17?Δgal2 ?Δagt1?Δstl1?Δmph2 ?Δmph3?leu2-3，112 ?ura3-52?trp1-289 ?his3-Δ1?MAL2-8 C?SUC2	Use 1% maltose as carbon source, strain growth; Be glucose, leucine, tryptophane and histidine auxotroph	P4H7GLUT4V 85M (selected marker URA3) ,=Seq ID No.3

Basic medium: 0.67% no amino acid yeast nitrogen (Difco); PH6.2.

Auxotrophy replenishes: leucine (0.44mM), tryptophane (0.19mM), Histidine (0.25mM), uridylic (0.44mM).Maltose can be 1-2%.

The international preservation of biomaterial proves

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15185
		The classification of organisms name	Yeast saccharomyces cerevisiae DSMefg
International depositary institution title	Germany microbial preservation center
		The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany

The international preservation survival of biomaterial proof

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15185
		International depositary institution title	Germany microbial preservation center
The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany
		Existing state	Survival

The international preservation of biomaterial proves

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15186
		The classification of organisms name	Yeast saccharomyces cerevisiae DSMhij
International depositary institution title	Germany microbial preservation center
		The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany

The international preservation survival of biomaterial proof

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15186
		International depositary institution title	Germany microbial preservation center
The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany
		Existing state	Survival

The international preservation of biomaterial proves

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15187
		The classification of organisms name	Yeast saccharomyces cerevisiae DSMsyz
International depositary institution title	Germany microbial preservation center
		The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany

The international preservation survival of biomaterial proof

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15187
		International depositary institution title	Germany microbial preservation center
The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany
		Existing state	Survival

The international preservation of biomaterial proves

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15188
		The classification of organisms name	Yeast saccharomyces cerevisiae DSMuvw
International depositary institution title	Germany microbial preservation center
		The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany

The international preservation survival of biomaterial proof

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15188
		International depositary institution title	Germany microbial preservation center
The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany
		Existing state	Survival

The international preservation of biomaterial proves

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15184
		The classification of organisms name	Yeast saccharomyces cerevisiae DSMdef
International depositary institution title	Germany microbial preservation center
		The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany

The international preservation survival of biomaterial proof

The preservation people	Aventis Pharma Deutschland GmbH
		Preservation day	On September 3rd, 2002
Preserving number	????DSM?15184
		International depositary institution title	Germany microbial preservation center
The preservation address	Mascheroder Weg 1b, D-38124 Brunswick, Germany
		Existing state	Survival

Sequence table

＜110〉Aventis Pharma Deutschland GmbH (Aventis Pharma Deutschland GmbH)

＜120〉purposes of yeast saccharomyces cerevisiae ERG4 mutant in the glucose transporter of expressing the Mammals source

<130>2002/0065

<140>

<141>

<150>10242763.1

<151>2002-09-14

<160>3

<170>PatentIn?Ver.2.1

<210>1

<211>1530

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

atgccgtcgg?gcttccaaca?gataggctcc?gaagatgggg?aaccccctca?gcagcgagtg?60

actgggaccc?tggtccttgc?tgtgttctct?gcggtgcttg?gctccctgca?gtttgggtac?120

aacattgggg?tcatcaatgc?ccctcagaag?gtgattgaac?agagctacaa?tgagacgtgg?180

ctggggaggc?aggggcctga?gggacccagc?tccatccctc?caggcaccct?caccaccctc?240

tgggccctct?ccatggccat?cttttccgtg?ggcggcatga?tttcctcctt?cctcattggt?300

atcatctctc?agtggcttgg?aaggaaaagg?gccatgctgg?tcaacaatgt?cctggcggtg?360

ctggggggca?gcctcatggg?cctggccaac?gctgctgcct?cctatgaaat?gctcatcctt?420

ggacgattcc?tcattggcgc?ctactcaggg?ctgacatcag?ggctggtgcc?catgtacgtg?480

ggggagattg?ctcccactca?cctgcggggc?gccctgggga?cgctcaacca?actggccatt?540

gttatcggca?ttctgatcgc?ccaggtgctg?ggcttggagt?ccctcctggg?cactgccagc?600

ctgtggccac?tgctcctggg?cctcacagtg?ctacctgccc?tcctgcagct?ggtcctgctg?660

cccttctgtc?ccgagagccc?ccgctacctc?tacatcatcc?agaatctcga?ggggcctgcc?720

agaaagagtc?tgaagcgcct?gacaggctgg?gccgatgttt?ctggagtgct?ggctgagctg?780

aaggatgaga?agcggaagct?ggagcgtgag?cggccactgt?ccctgctcca?gctcctgggc?840

agccgtaccc?accggcagcc?cctgatcatt?gcggtcgtgc?tgcagctgag?ccagcagctc?900

tctggcatca?atgctgtttt?ctattattcg?accagcatct?tcgagacagc?aggggtaggc?960

cagcctgcct?atgccaccat?aggagctggt?gtggtcaaca?cagtcttcac?cttggtctcg?1020

gtgttgttgg?tggagcgggc?ggggcgccgg?acgctccatc?tcctgggcct?ggcgggcatg?1080

tgtggctgtg?ccatcctgat?gactgtggct?ctgctcctgc?tggagcgagt?tccagccatg?1140

agctacgtct?ccattgtggc?catctttggc?ttcgtggcat?tttttgagat?tggccctggc?1200

cccattcctt?ggttcatcgt?ggccgagctc?ttcagccagg?gaccccgccc?ggcagccatg?1260

gctgtggctg?gtttctccaa?ctggacgagc?aacttcatca?ttggcatggg?tttccagtat?1320

gttgcggagg?ctatggggcc?ctacgtcttc?cttctatttg?cggtcctcct?gctgggcttc?1380

ttcatcttca?ccttcttaag?agtacctgaa?actcgaggcc?ggacgtttga?ccagatctca?1440

gctgccttcc?accggacacc?ctctctttta?gagcaggagg?tgaaacccag?cacagaactt?1500

gagtatttag?ggccagatga?gaacgactga??????????????????????????????????1530

<210>2

<211>509

<212>PRT

＜213〉homo sapiens

<400>2

Met?Pro?Ser?Gly?Phe?Gln?Gln?Ile?Gly?Ser?Glu?Asp?Gly?Glu?Pro?Pro

1???????????????5???????????????????10??????????????????15

Gln?Gln?Arg?Val?Thr?Gly?Thr?Leu?Val?Leu?Ala?Val?Phe?Ser?Ala?Val

20???????????????????25?????????????????30

Leu?Gly?Ser?Leu?Gln?Phe?Gly?Tyr?Asn?Ile?Gly?Val?Ile?Asn?Ala?Pro

35??????????????????40??????????????????45

Gln?Lys?Val?Ile?Glu?Gln?Ser?Tyr?Asn?Glu?Thr?Trp?Leu?Gly?Arg?Gln

50??????????????????55??????????????????60

Gly?Pro?Glu?Gly?Pro?Ser?Set?Ile?Pro?Pro?Gly?Thr?Leu?Thr?Thr?Leu

65??????????????????70??????????????????75??????????????????80

Trp?Ala?Leu?Ser?Met?Ala?Ile?Phe?Ser?Val?Gly?Gly?Met?Ile?Ser?Ser

85??????????????????90??????????????????95

Phe?Leu?Ile?Gly?Ile?Ile?Ser?Gln?Trp?Leu?Gly?Arg?Lys?Arg?Ala?Met

100?????????????????105?????????????????110

Leu?Val?Asn?Asn?Val?Leu?Ala?Val?Leu?Gly?Gly?Ser?Leu?Met?Gly?Leu

115?????????????????120?????????????????125

Ala?Asn?Ala?Ala?Ala?Ser?Tyr?Glu?Met?Leu?Ile?Leu?Gly?Arg?Phe?Leu

130?????????????????135?????????????????140

Ile?Gly?Ala?Tyr?Ser?Gly?Leu?Thr?Ser?Gly?Leu?Val?Pro?Met?Tyr?Val

145?????????????????150?????????????????155?????????????????160

Gly?Glu?Ile?Ala?Pro?Thr?His?Leu?Arg?Gly?Ala?Leu?Gly?Thr?Leu?Asn

165?????????????????170?????????????????175

Gln?Leu?Ala?Ile?Val?Ile?Gly?Ile?Leu?Ile?Ala?Gln?Val?Leu?Gly?Leu

180?????????????????185?????????????????190

Glu?Ser?Leu?Leu?Gly?Thr?Ala?Ser?Leu?Trp?Pro?Leu?Leu?Leu?Gly?Leu

195?????????????????200?????????????????205

Thr?Val?Leu?Pro?Ala?Leu?Leu?Gln?Leu?Val?Leu?Leu?Pro?Phe?Cys?Pro

210?????????????????215?????????????????220

Glu?Ser?Pro?Arg?Tyr?Leu?Tyr?Ile?Ile?Gln?Asn?Leu?Glu?Gly?Pro?Ala

225?????????????????230?????????????????235?????????????????240

Arg?Lys?Ser?Leu?Lys?Arg?Leu?Thr?Gly?Trp?Ala?Asp?Val?Ser?Gly?Val

245?????????????????250?????????????????255

Leu?Ala?Glu?Leu?Lys?Asp?Glu?Lys?Arg?Lys?Leu?Glu?Arg?Glu?Arg?Pro

260?????????????????265?????????????????270

Leu?Ser?Leu?Leu?Gln?Leu?Leu?Gly?Ser?Arg?Thr?His?Arg?Gln?Pro?Leu

275?????????????????280?????????????????285

Ile?Ile?Ala?Val?Val?Leu?Gln?Leu?Ser?Gln?Gln?Leu?Ser?Gly?Ile?Asn

290?????????????????295?????????????????300

Ala?Val?Phe?Tyr?Tyr?Ser?Thr?Ser?Ile?Phe?Glu?Thr?Ala?Gly?Val?Gly

305?????????????????310?????????????????315?????????????????320

Gln?Pro?Ala?Tyr?Ala?Thr?Ile?Gly?Ala?Gly?Val?Val?Asn?Thr?Val?Phe

325?????????????????330?????????????????335

Thr?Leu?Val?Ser?Val?Leu?Leu?Val?Glu?Arg?Ala?Gly?Arg?Arg?Thr?Leu

340?????????????????345?????????????????350

His?Leu?Leu?Gly?Leu?Ala?Gly?Met?Cys?Gly?Cys?Ala?Ile?Leu?Met?Thr

355?????????????????360?????????????????365

Val?Ala?Leu?Leu?Leu?Leu?Glu?Arg?Val?Pro?Ala?Met?Ser?Tyr?Val?Ser

370?????????????????375?????????????????380

Ile?Val?Ala?Ile?Phe?Gly?Phe?Val?Ala?Phe?Phe?Glu?Ile?Gly?Pro?Gly

385?????????????????390?????????????????395?????????????????400

Pro?Ile?Pro?Trp?Phe?Ile?Val?Ala?Glu?Leu?Phe?Ser?Gln?Gly?Pro?Arg

405?????????????????410?????????????????415

Pro?Ala?Ala?Met?Ala?Val?Ala?Gly?Phe?Ser?Asn?Trp?Thr?Ser?Asn?Phe

420?????????????????425?????????????????430

Ile?Ile?Gly?Met?Gly?Phe?Gln?Tyr?Val?Ala?Glu?Ala?Met?Gly?Pro?Tyr

435?????????????????440?????????????????445

Val?Phe?Leu?Leu?Phe?Ala?Val?Leu?Leu?Leu?Gly?Phe?Phe?Ile?Phe?Thr

450?????????????????455?????????????????460

Phe?Leu?Arg?Val?Pro?Glu?Thr?Arg?Gly?Arg?Thr?Phe?Asp?Gln?Ile?Ser

465?????????????????470?????????????????475?????????????????480

Ala?Ala?Phe?His?Arg?Thr?Pro?Ser?Leu?Leu?Glu?Gln?Glu?Val?Lys?Pro

485?????????????????490?????????????????495

Ser?Thr?Glu?Leu?Glu?Tyr?Leu?Gly?Pro?Asp?Glu?Asn?Asp

500?????????????????505

<210>3

<211>7809

<212>DNA

＜213〉yeast saccharomyces cerevisiae

<400>3

cgtaggaaca?atttcgggcc?cctgcgtgtt?cttctgaggt?tcatctttta?catttgcttc?60

tgctggataa?ttttcagagg?caacaaggaa?aaattagatg?gcaaaaagtc?gtctttcaag?120

gaaaaatccc?caccatcttt?cgagatcccc?tgtaacttat?tggcaactga?aagaatgaaa?180

aggaggaaaa?tacaaaatat?actagaactg?aaaaaaaaaa?agtataaata?gagacgatat?240

atgccaatac?ttcacaatgt?tcgaatctat?tcttcatttg?cagctattgt?aaaataataa?300

aacatcaaga?acaaacaagc?tcaacttgtc?ttttctaaga?acaaagaata?aacacaaaaa?360

caaaaagttt?ttttaatttt?aatcaaaaaa?tgccgtcggg?cttccaacag?ataggctccg?420

aagatgggga?accccctcag?cagcgagtga?ctgggaccct?ggtccttgct?gtgttctctg?480

cggtgcttgg?ctccctgcag?tttgggtaca?acattggggt?catcaatgcc?cctcagaagg?540

tgattgaaca?gagctacaat?gagacgtggc?tggggaggca?ggggcctgag?ggacccagct?600

ccatccctcc?aggcaccctc?accaccctct?gggccctctc?catggccatc?ttttccgtgg?660

gcggcatgat?ttcctccttc?ctcattggta?tcatctctca?gtggcttgga?aggaaaaggg?720

ccatgctggt?caacaatgtc?ctggcggtgc?tggggggcag?cctcatgggc?ctggccaacg?780

ctgctgcctc?ctatgaaatg?ctcatccttg?gacgattcct?cattggcgcc?tactcagggc?840

tgacatcagg?gctggtgccc?atgtacgtgg?gggagattgc?tcccactcac?ctgcggggcg?900

ccctggggac?gctcaaccaa?ctggccattg?ttatcggcat?tctgatcgcc?caggtgctgg?960

gcttggagtc?cctcctgggc?actgccagcc?tgtggccact?gctcctgggc?ctcacagtgc?1020

tacctgccct?cctgcagctg?gtcctgctgc?ccttctgtcc?cgagagcccc?cgctacctct?1080

acatcatcca?gaatctcgag?gggcctgcca?gaaagagtct?gaagcgcctg?acaggctggg?1140

ccgatgtttc?tggagtgctg?gctgagctga?aggatgagaa?gcggaagctg?gagcgtgagc?1200

ggccactgtc?cctgctccag?ctcctgggca?gccgtaccca?ccggcagccc?ctgatcattg?1260

cggtcgtgct?gcagctgagc?cagcagctct?ctggcatcaa?tgctgttttc?tattattcga?1320

ccagcatctt?cgagacagca?ggggtaggcc?agcctgccta?tgccaccata?ggagctggtg?1380

tggtcaacac?agtcttcacc?ttggtctcgg?tgttgttggt?ggagcgggcg?gggcgccgga?1440

cgctccatct?cctgggcctg?gcgggcatgt?gtggctgtgc?catcctgatg?actgtggctc?1500

tgctcctgct?ggagcgagtt?ccagccatga?gctacgtctc?cattgtggcc?atctttggct?1560

tcgtggcatt?ttttgagatt?ggccctggcc?ccattccttg?gttcatcgtg?gccgagctct?1620

tcagccaggg?accccgcccg?gcagccatgg?ctgtggctgg?tttctccaac?tggacgagca?1680

acttcatcat?tggcatgggt?ttccagtatg?ttgcggaggc?tatggggccc?tacgtcttcc?1740

ttctatttgc?ggtcctcctg?ctgggcttct?tcatcttcac?cttcttaaga?gtacctgaaa?1800

ctcgaggccg?gacgtttgac?cagatctcag?ctgccttcca?ccggacaccc?tctcttttag?1860

agcaggaggt?gaaacccagc?acagaacttg?agtatttagg?gccagatgag?aacgactgac?1920

tcgagtcatg?taattagtta?tgtcacgctt?acattcacgc?cctcccccca?catccgctct?1980

aaccgaaaag?gaaggagtta?gacaacctga?agtctaggtc?cctatttatt?tttttatagt?2040

tatgttagta?ttaagaacgt?tatttatatt?tcaaattttt?cttttttttc?tgtacagacg?2100

cgtgtacgca?tgtaacatta?tactgaaaac?cttgcttgag?aaggttttgg?gacgctcgaa?2160

ggctttaatt?tgcggccggt?acccaattcg?ccctatagtg?agtcgtatta?cgcgcgctca?2220

ctggccgtcg?ttttacaacg?tcgtgactgg?gaaaaccctg?gcgttaccca?acttaatcgc?2280

cttgcagcac?atcccccttt?cgccagctgg?cgtaatagcg?aagaggcccg?caccgatcgc?2340

ccttcccaac?agttgcgcag?cctgaatggc?gaatggcgcg?acgcgccctg?tagcggcgca?2400

ttaagcgcgg?cgggtgtggt?ggttacgcgc?agcgtgaccg?ctacacttgc?cagcgcccta?2460

gcgcccgctc?ctttcgcttt?cttcccttcc?tttctcgcca?cgttcgccgg?ctttccccgt?2520

caagctctaa?atcgggggct?ccctttaggg?ttccgattta?gtgctttacg?gcacctcgac?2580

cccaaaaaac?ttgattaggg?tgatggttca?cgtagtgggc?catcgccctg?atagacggtt?2640

tttcgccctt?tgacgttgga?gtccacgttc?tttaatagtg?gactcttgtt?ccaaactgga?2700

acaacactca?accctatctc?ggtctattct?tttgatttat?aagggatttt?gccgatttcg?2760

gcctattggt?taaaaaatga?gctgatttaa?caaaaattta?acgcgaattt?taacaaaata?2820

ttaacgttta?caatttcctg?atgcggtatt?ttctccttac?gcatctgtgc?ggtatttcac?2880

accgcatagg?gtaataactg?atataattaa?attgaagctc?taatttgtga?gtttagtata?2940

catgcattta?cttataatac?agttttttag?ttttgctggc?cgcatcttct?caaatatgct?3000

tcccagcctg?cttttctgta?acgttcaccc?tctaccttag?catcccttcc?ctttgcaaat?3060

agtcctcttc?caacaataat?aatgtcagat?cctgtagaga?ccacatcatc?cacggttcta?3120

tactgttgac?ccaatgcgtc?tcccttgtca?tctaaaccca?caccgggtgt?cataatcaac?3180

caatcgtaac?cttcatctct?tccacccatg?tctctttgag?caataaagcc?gataacaaaa?3240

tctttgtcgc?tcttcgcaat?gtcaacagta?cccttagtat?attctccagt?agatagggag?3300

cccttgcatg?acaattctgc?taacatcaaa?aggcctctag?gttcctttgt?tacttcttct?3360

gccgcctgct?tcaaaccgct?aacaatacct?gggcccacca?caccgtgtgc?attcgtaatg?3420

tctgcccatt?ctgctattct?gtatacaccc?gcagagtact?gcaatttgac?tgtattacca?3480

atgtcagcaa?attttctgtc?ttcgaagagt?aaaaaattgt?acttggcgga?taatgccttt?3540

agcggcttaa?ctgtgccctc?catggaaaaa?tcagtcaaga?tatccacatg?tgtttttagt?3600

aaacaaattt?tgggacctaa?tgcttcaact?aactccagta?attccttggt?ggtacgaaca?3660

tccaatgaag?cacacaagtt?tgtttgcttt?tcgtgcatga?tattaaatag?cttggcagca?3720

acaggactag?gatgagtagc?agcacgttcc?ttatatgtag?ctttcgacat?gatttatctt?3780

cgtttcctgc?aggtttttgt?tctgtgcagt?tgggttaaga?atactgggca?atttcatgtt?3840

tcttcaacac?tacatatgcg?tatatatacc?aatctaagtc?tgtgctcctt?ccttcgttct?3900

tccttctgtt?cggagattac?cgaatcaaaa?aaatttcaaa?gaaaccgaaa?tcaaaaaaaa?3960

gaataaaaaa?aaaatgatga?attgaattga?aaagctgtgg?tatggtgcac?tctcagtaca?4020

atctgctctg?atgccgcata?gttaagccag?ccccgacacc?cgccaacacc?cgctgacgcg?4080

ccctgacggg?cttgtctgct?cccggcatcc?gcttacagac?aagctgtgac?cgtctccggg?4140

agctgcatgt?gtcagaggtt?ttcaccgtca?tcaccgaaac?gcgcgagacg?aaagggcctc?4200

gtgatacgcc?tatttttata?ggttaatgtc?atgataataa?tggtttctta?gtatgatcca?4260

atatcaaagg?aaatgatagc?attgaaggat?gagactaatc?caattgagga?gtggcagcat?4320

atagaacagc?taaagggtag?tgctgaagga?agcatacgat?accccgcatg?gaatgggata?4380

atatcacagg?aggtactaga?ctacctttca?tcctacataa?atagacgcat?ataagtacgc?4440

atttaagcat?aaacacgcac?tatgccgttc?ttctcatgta?tatatatata?caggcaacac?4500

gcagatatag?gtgcgacgtg?aacagtgagc?tgtatgtgcg?cagctcgcgt?tgcattttcg?4560

gaagcgctcg?ttttcggaaa?cgctttgaag?ttcctattcc?gaagttccta?ttctctagaa?4620

agtataggaa?cttcagagcg?cttttgaaaa?ccaaaagcgc?tctgaagacg?cactttcaaa?4680

aaaccaaaaa?cgcaccggac?tgtaacgagc?tactaaaata?ttgcgaatac?cgcttccaca?4740

aacattgctc?aaaagtatct?ctttgctata?tatctctgtg?ctatatccct?atataaccta?4800

cccatccacc?tttcgctcct?tgaacttgca?tctaaactcg?acctctacat?tttttatgtt?4860

tatctctagt?attactcttt?agacaaaaaa?attgtagtaa?gaactattca?tagagtgaat?4920

cgaaaacaat?acgaaaatgt?aaacatttcc?tatacgtagt?atatagagac?aaaatagaag?4980

aaaccgttca?taattttctg?accaatgaag?aatcatcaac?gctatcactt?tctgttcaca?5040

aagtatgcgc?aatccacatc?ggtatagaat?ataatcgggg?atgcctttat?cttgaaaaaa?5100

tgcacccgca?gcttcgctag?taatcagtaa?acgcgggaag?tggagtcagg?ctttttttat?5160

ggaagagaaa?atagacacca?aagtagcctt?cttctaacct?taacggacct?acagtgcaaa?5220

aagttatcaa?gagactgcat?tatagagcgc?acaaaggaga?aaaaaagtaa?tctaagatgc?5280

tttgttagaa?aaatagcgct?ctcgggatgc?atttttgtag?aacaaaaaag?aagtatagat?5340

tctttgttgg?taaaatagcg?ctctcgcgtt?gcatttctgt?tctgtaaaaa?tgcagctcag?5400

attctttgtt?tgaaaaatta?gcgctctcgc?gttgcatttt?tgttttacaa?aaatgaagca?5460

cagattcttc?gttggtaaaa?tagcgctttc?gcgttgcatt?tctgttctgt?aaaaatgcag?5520

ctcagattct?ttgtttgaaa?aattagcgct?ctcgcgttgc?atttttgttc?tacaaaatga?5580

agcacagatg?cttcgttcag?gtggcacttt?tcggggaaat?gtgcgcggaa?cccctatttg?5640

tttatttttc?taaatacatt?caaatatgta?tccgctcatg?agacaataac?cctgataaat?5700

gcttcaataa?tattgaaaaa?ggaagagtat?gagtattcaa?catttccgtg?tcgcccttat?5760

tccctttttt?gcggcatttt?gccttcctgt?ttttgctcac?ccagaaacgc?tggtgaaagt?5820

aaaagatgct?gaagatcagt?tgggtgcacg?agtgggttac?atcgaactgg?atctcaacag?5880

cggtaagatc?cttgagagtt?ttcgccccga?agaacgtttt?ccaatgatga?gcacttttaa?5940

agttctgcta?tgtggcgcgg?tattatcccg?tattgacgcc?gggcaagagc?aactcggtcg?6000

ccgcatacac?tattctcaga?atgacttggt?tgagtactca?ccagtcacag?aaaagcatct?6060

tacggatggc?atgacagtaa?gagaattatg?cagtgctgcc?ataaccatga?gtgataacac?6120

tgcggccaac?ttacttctga?caacgatcgg?aggaccgaag?gagctaaccg?cttttttgca?6180

caacatgggg?gatcatgtaa?ctcgccttga?tcgttgggaa?ccggagctga?atgaagccat?6240

accaaacgac?gagcgtgaca?ccacgatgcc?tgtagcaatg?gcaacaacgt?tgcgcaaact?6300

attaactggc?gaactactta?ctctagcttc?ccggcaacaa?ttaatagact?ggatggaggc?6360

ggataaagtt?gcaggaccac?ttctgcgctc?ggcccttccg?gctggctggt?ttattgctga?6420

taaatctgga?gccggtgagc?gtgggtctcg?cggtatcatt?gcagcactgg?ggccagatgg?6480

taagccctcc?cgtatcgtag?ttatctacac?gacggggagt?caggcaacta?tggatgaacg?6540

aaatagacag?atcgctgaga?taggtgcctc?actgattaag?cattggtaac?tgtcagacca?6600

agtttactca?tatatacttt?agattgattt?aaaacttcat?ttttaattta?aaaggatcta?6660

ggtgaagatc?ctttttgata?atctcatgac?caaaatccct?taacgtgagt?tttcgttcca?6720

ctgagcgtca?gaccccgtag?aaaagatcaa?aggatcttct?tgagatcctt?tttttctgcg?6780

cgtaatctgc?tgcttgcaaa?caaaaaaacc?accgctacca?gcggtggttt?gtttgccgga?6840

tcaagagcta?ccaactcttt?ttccgaaggt?aactggcttc?agcagagcgc?agataccaaa?6900

tactgtcctt?ctagtgtagc?cgtagttagg?ccaccacttc?aagaactctg?tagcaccgcc?6960

tacatacctc?gctctgctaa?tcctgttacc?agtggctgct?gccagtggcg?ataagtcgtg?7020

tcttaccggg?ttggactcaa?gacgatagtt?accggataag?gcgcagcggt?cgggctgaac?7080

ggggggttcg?tgcacacagc?ccagcttgga?gcgaacgacc?tacaccgaac?tgagatacct?7140

acagcgtgag?ctatgagaaa?gcgccacgct?tcccgaaggg?agaaaggcgg?acaggtatcc?7200

ggtaagcggc?agggtcggaa?caggagagcg?cacgagggag?cttccagggg?gaaacgcctg?7260

gtatctttat?agtcctgtcg?ggtttcgcca?cctctgactt?gagcgtcgat?ttttgtgatg?7320

ctcgtcaggg?gggcggagcc?tatggaaaaa?cgccagcaac?gcggcctttt?tacggttcct?7380

ggccttttgc?tggccttttg?ctcacatgtt?ctttcctgcg?ttatcccctg?attctgtgga?7440

taaccgtatt?accgcctttg?agtgagctga?taccgctcgc?cgcagccgaa?cgaccgagcg?7500

cagcgagtca?gtgagcgagg?aagcggaaga?gcgcccaata?cgcaaaccgc?ctctccccgc?7560

gcgttggccg?attcattaat?gcagctggca?cgacaggttt?cccgactgga?aagcgggcag?7620

tgagcgcaac?gcaattaatg?tgagttacct?cactcattag?gcaccccagg?ctttacactt?7680

tatgcttccg?gctcctatgt?tgtgtggaat?tgtgagcgga?taacaatttc?acacaggaaa?7740

cagctatgac?catgattacg?ccaagcgcgc?aattaaccct?cactaaaggg?aacaaaagct?7800

ggagctttt?????????????????????????????????????????????????????????7809

Claims (36)

1. purifying and isolating polynucleotide, it contains coding GLUT4V85M protein DNA sequence.

2. the polynucleotide of claim 1, it contains from arbitrary sequence of group down:

A) according to the nucleotide sequence of Seq ID No.1

B) under stringent condition with Seq ID No.1 sequence hybridization and the proteinic nucleotide sequence of the GLUT4V85M that encodes.

3. claim 1 or 2 polynucleotide, wherein GLUT4V85M protein has the aminoacid sequence according to Seq ID No.2.

4. the polynucleotide of claim 1 to 3, wherein the proteinic coding region of GLUT4V85M is connected with the promotor operability.

5. the polynucleotide of claim 1 to 4, it can duplicate in yeast cell.

6. the polynucleotide of claim 5, it is used in marking protein in the yeast cell.

7. from the yeast cell of yeast saccharomyces cerevisiae, wherein all glucose transporter no longer have function and do not contain functional Erg4 protein.

8. from the yeast cell of yeast saccharomyces cerevisiae, wherein all glucose transporter no longer have function and do not contain functional Fgy1 protein and functional Erg4 protein.

9. claim 7 or 8 yeast cell, wherein the ERG4 gene is lacked wholly or in part.

10. the yeast cell of claim 7, it is as yeast saccharomyces cerevisiae DSM 15187 preservations.

11. the yeast cell of claim 8 or 9, it is as yeast saccharomyces cerevisiae DSM 15184 preservations.

12. the purposes of the yeast cell of claim 15 to 18 in expressing Mammals GLUT1 protein or GLUT4 protein.

13. the purposes of claim 12 is used for expressing human GLUT1 protein or people GLUT4 protein.

14. the yeast cell of claim 7, it contains the polynucleotide of claim 1 to 6.

15. the yeast cell of claim 14, it contains GLUT4V85M protein.

16. the yeast cell of claim 14 or 15, it is as yeast saccharomyces cerevisiae DSM 15185 preservations.

17. the method for the yeast cell of preparation claim 14 to 16, it comprises step:

A) provide the yeast cell of claim 7,

B) provide the polynucleotide of claim 5 or 6,

C) use b) polynucleotide transform a) yeast cell,

D) select transformed yeast cells,

E) express GLUT4V85M protein suitably the time.

18. the yeast cell of claim 8 or 9, it contains the polynucleotide of claim 1 to 6.

19. the yeast cell of claim 18, it contains GLUT4V85M protein.

20. the yeast cell of claim 18 or 19, it is as yeast saccharomyces cerevisiae DSM 15186 preservations.

21. the method for the yeast cell of preparation claim 18 to 20, it comprises step:

A) provide the yeast cell of claim 8 or 9,

B) provide the polynucleotide of claim 5 or 6,

C) use b) polynucleotide transform a) yeast cell,

D) select transformed yeast cells,

E) express GLUT4V85M protein suitably the time.

22. its whole glucose transporter no longer have the yeast cell of function, it contains the polynucleotide of claim 1 to 6.

23. the yeast cell of claim 22, it contains GLUT4V85M protein.

24. the yeast cell of claim 22 or 23, it is as yeast saccharomyces cerevisiae DSM 15188 preservations.

25. the method for the yeast cell of preparation claim 22 to 24, it comprises step:

A) produce the yeast cell that its whole glucose transporter no longer have function,

B) provide the polynucleotide of claim 5 or 6,

C) use b) polynucleotide transform a) yeast cell,

D) select transformed yeast cells,

E) express GLUT4V85M protein suitably the time.

26. have the protein of the functionally active of glucose transporter, it is by each polynucleotide sequence coding in the claim 1 to 3.

27. the protein of claim 13, it contains the aminoacid sequence of with good grounds Seq ID No.2.

28. identify the method for the compound that stimulates the GLUT4 protein active, it comprises step:

A) provide one or multinomial yeast cell in the claim 14 to 17,

B) provide chemical compound,

C) with a) yeast and b) chemical compound contact,

D) zymic glucose uptake mensuration c),

E) with d) the glucose uptake detected value and not with b) described in chemical compound contacted a) described in the glucose uptake detected value of yeast cell compare, cause d) described in the compound that increases of zymic glucose uptake amount stimulate described GLUT4 activity of proteins.

29. contain method institute's compounds identified of claim 28 and be used for the additive of compounding pharmaceutical and the medicine of vehicle.

30. the method for claim 28 institute compounds identified is used for the treatment of purposes in the medicine of I type and/or type ii diabetes in preparation.

31. identify the method for the compound of the respective egg white matter that suppresses the Fgy1 gene, it comprises step:

A) provide one or multinomial yeast cell in the claim 7 to 10, this yeast cell contains GLUT4 protein,

B) provide chemical compound,

C) with a) yeast and b) chemical compound contact,

D) zymic glucose uptake mensuration c),

E) with d) the glucose uptake detected value and not with b) described in chemical compound contacted a) described in the glucose uptake detected value of yeast cell compare, cause d) described in the compound that increases of zymic glucose uptake amount suppress the Fgy1 activity of proteins.

32. contain method institute's compounds identified of claim 31 and be used for the additive of compounding pharmaceutical and the medicine of vehicle.

33. the method for claim 31 institute compounds identified is used for the treatment of purposes in the medicine of diabetes in preparation.

34. identify the method for the proteinic compound that suppresses the ERG4 genes encoding, it comprises step:

A) provide one or multinomial yeast cell in the claim 22 to 25,

B) provide chemical compound,

C) with a) yeast and b) chemical compound contact,

D) zymic glucose uptake mensuration c),

E) with d) the glucose uptake detected value and not with b) described in chemical compound contacted a) described in the glucose uptake detected value of yeast cell compare, cause d) described in the compound that increases of zymic glucose uptake amount suppress the Erg4 activity of proteins.

35. contain method institute's compounds identified of claim 34 and be used for the additive of compounding pharmaceutical and the medicine of vehicle.

36. the method for claim 34 institute compounds identified is used for the treatment of purposes in the medicine of diabetes in preparation.