US20120156695A1 - Microorganism for expressing a human membrane protein - Google Patents

Microorganism for expressing a human membrane protein Download PDF

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US20120156695A1
US20120156695A1 US13/321,371 US201013321371A US2012156695A1 US 20120156695 A1 US20120156695 A1 US 20120156695A1 US 201013321371 A US201013321371 A US 201013321371A US 2012156695 A1 US2012156695 A1 US 2012156695A1
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organism
protein
precursor
saccharomyces
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Michael Schilling
Christine Lang
Andreas Raab
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Novozymes AS
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OrganoBalance GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention relates to an isolated genetically modified living non-mammal organism, having increased HMG-CoA reductase activity compared to the wild type, and having a reduced C24 methyltransferase and/or delta22 desaturase activity compared to the wild type, to the uses thereof, a nucleic acid construct for the production thereof, a kit containing the organism, and a membrane extract of such an organism.
  • membrane proteins in particular human membrane proteins, functionally in a native conformation in at least analytically well acceptable amounts or to make systems available that contain these proteins in a functionally active manner in an amount being for instance sufficient for screening purposes.
  • biosynthesis methods by means of microorganisms can be used for this.
  • the heterologous expression of membrane proteins from mammal cells in wild type strains often involves difficulties because of the lack of cholesterol, or the presence of ergosterol, respectively, since the expressed membrane proteins are often functionally limited or inactive. This can be explained by the well known fact that sterols in the membranes play an important role for correct folding and the induction of an active conformation of membrane proteins and thus their functionality, i.e. for a functional expression of heterologous membrane proteins in a yeast, the sterol pattern of the membranes must match the membrane protein.
  • Sterols are an essential constituent of the membranes of eukaryotic cells. They are responsible for the fluidity and permeability of the membranes. Particularly noteworthy is their contribution to the regulation of numerous membrane proteins. They are important as a kind of cofactors for correct folding, stability and activity of the membrane proteins. Free sterols can be found in eukaryotic cells in the plasma membrane and the membranes of all cell compartments. In the lipid particles, sterols occur in an esterified form as storage lipids. In yeasts, the largest part of the free sterols is located in the plasma membrane, followed by the secretory vesicles, and the amount in microsomes, vacuoles and mitochondrial membranes is small.
  • Ergosterol is the final product of the sterol biosynthesis pathway for instance in the yeast S. cerevisiae and is the main sterol in the plasma membrane and the secretory vesicles.
  • the membranes of the subcellular compartments contain smaller amounts of sterols, but also other sterol intermediates.
  • the final product of the sterol biosynthesis is cholesterol, which is the largest part of the sterols in the plasma membrane.
  • the plasma membrane contains approx. 60-80% of the total cellular cholesterol, the ER, the place of the sterol synthesis, contains only approx. 0.5-1%.
  • humanization relates, however, to the glycosylation pattern of heterologously produced proteins, which has been adapted to human protein, and not to the sterol pattern being relevant for the present invention. Mainly, the synthesis of enzymes or antibodies is treated, and not the one of membrane proteins.
  • the ergosterol biosynthesis pathway of yeasts can be subdivided into the pre-squalene pathway, i.e. the synthesis of squalene from acetyl-CoA molecules, and the post-squalene pathway, in which the reaction of squalene to ergosterol is catalyzed.
  • the hydroxy-methyl-glutaryl coenzyme A reductase HMG-CoA reductase
  • the high energy-consuming ergosterol biosynthesis pathway is regulated mainly by this enzyme, which is subject therefore to numerous regulation mechanisms, such as e.g. the feedback inhibition by ergosterol.
  • the differences downstream of zymosterol to ergosterol or cholesterol are explained in the following.
  • yeast cells zymosterol is reacted by C24 methyltransferase to fecosterol, then by delta7-delta8 isomerase to episterol, by delta5 desaturase to ergosta-5,7,24(28)-trienol, by delta22 desaturase to ergosta-5,7,22,24(28)-tetraenol and finally by delta24 reductase to ergosterol.
  • zymosterol is reacted by delta7-delta8 isomerase to cholesta-7,24-dienol, by delta5 desaturase to cholesta-5,7,24-trienol and by delta7 reductase to desmosterol, and finally by sterol-delta5 desaturase to cholesterol.
  • the last-mentioned enzyme can also react cholesta-7,24-dienol to lathosterol and cholesta-5,7,24-trienol to 7-dehydrocholesterol.
  • Lathosterol in turn can be reacted by delta5 desaturase to 7-dehydrocholesterol. The latter in turn reacts by delta7 reductase also to cholesterol.
  • SERT serotonin transporter
  • the serotonin transporter belongs to the family of the Na + /Cl ⁇ -dependent neurotransmitter transporters, which are responsible for the re-uptake of biogenic amines from the synaptic space back to the presynaptic neurons. Further members of this family are the transporters for noradrenaline, dopamine, choline, glycine and ⁇ -aminobutyric acid.
  • the serotonin transporter has a high clinical importance as a pharmacological target molecule of many antidepressants.
  • the antidepressants, the target of which is the SERT can be subdivided into two main groups: On the one hand the tricyclic agent molecules, such as e.g.
  • SSRI's selective serotonin re-uptake inhibitors.
  • active agents such as e.g. paroxetine, fluoxetine, sertraline and citalopram.
  • the SERT was cloned from the human, rat, mouse, bovine and fruit fly Drosophila melanogaster tissue.
  • the SERT was expressed in all common systems, i.e. in E. coli and the yeast Pichia pastoris , in insect cells and in different cell lines, such as for instance COS-1 cells, BHK cells, Hela cells and HEK cells.
  • An expression of the SERT in the yeast Saccharomyces cerevisiae is not known.
  • a functional expression could only be achieved in mammal cell lines, and in insect cells, not however in E. coli or Pichia pastoris .
  • the SERT is an extremely difficult protein with regard to heterologous or overexpression.
  • a modified organism for instance a strain of the yeast Saccharomyces cerevisiae , which is capable to synthesize cholesterol or a pre-stage, which can compensate the lack of cholesterol with regard to the serotonin transporter, would for the first time give point to making possible a functional expression of this protein in non-mammals.
  • a functional expression such an organism could be used as a basis for the design of a “bioassay”. With this “bioassay”, novel pharmacologically relevant active agents for the serotonin transporter could be identified by the “screening” different substance groups.
  • yeast strain could also be used for the functional expression of other cholesterol-dependent membrane proteins.
  • FIG. 1 shows a diagrammatic representation of the episomal yeast expression plasmids pFlat1 and pFlat3.
  • FIG. 2 shows a scintillation count of the bound [3H] citalopram in fmol per 109 cells.
  • FIG. 2 further shows that the non-specific binding is highest for GRFcol-DHCR7 and lowest for GRFura3 anaerobic+cholesterol.
  • FIG. 3 shows the gene sequence for suitable HMG-CoA reductases such as tHMG1.
  • the invention teaches inter alia an isolated genetically modified living non-mammal organism, having an increased HMG-CoA reductase activity compared to the wild type, and having a reduced C24 methyltransferase and/or delta22 desaturase activity compared to the wild type, wherein the organism has an increased dehydrocholesterol-delta7 reductase activity compared to the wild type.
  • the invention is based on the surprising finding that for a functional expression of mammal membrane proteins, i.e. the expression in a folding, which promotes or even at all allows the enzymatic activity of the membrane protein, not only the presence of cholesterol is useful or necessary, but that rather the same success can already be achieved with the presence of desmosterol. Further it has been found that the desmosterol is in fact also located in the (plasma) membrane of the organism. That means that a non-mammal organism is created that has, compared to the wild type, in a membrane, in particular the plasma membrane, an increased content of desmosterol, and if applicable, as will be explained below, of cholesterol.
  • mammal membrane proteins in particular human membrane proteins
  • the membrane proteins can thus be functionally produced in high quantities and in a simple and economical way. This can be further used in manifold ways.
  • an organism according to the invention can for instance also be used for identifying substances binding to the (functional) membrane protein, for instance by screening.
  • An organism according to the invention is therefore a universal instrument for the functional synthesis of virtually any membrane proteins, for instance plasma membrane proteins, from mammals, such as humans, and for finding agonists and/or antagonists of such membrane proteins.
  • the invention has therefore also special importance as an instrument for finding pharmaceutically suitable substances.
  • the organism additionally has an increased dehydrocholesterol-delta24 reductase activity compared to the wild type. Thereby occurs a further reaction of the desmosterol to cholesterol as well as of the desmosterol precursors cholesta-7,24-dienol and cholesta-5,7,24-trienol to the cholesterol precursors lathosterol and 7-dehydrocholesterol.
  • the organism has an increased squalene epoxidase and/or lanosterol demethylase activity compared to the wild type. Thereby, the biosynthesis of zymosterol from squalene is promoted and therefore also the production of desmosterol or cholesterol.
  • the organism may be a prokaryotic organism, in particular selected from the group consisting of bacteria of the species Bacillus, Escherichia, Lactobacillus, Corynebacterium, Acetobacter, Acinetobacter and Pseudomonas , or a eukaryotic organism, in particular selected from the group consisting of algae of the species Cryptista, Chloromonadophyceae, Xanthophyceae, Crypthecodinium, Chrysophyta, Bacillariophyta, Phaeophyta, Rhodophyta, Chlorophyta, Haptophyta, Cryptista, Euglenozoa, Dinozoa, Chlorarachniophyta , yeasts, insect cells from Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, Drosophila , and
  • yeast it may be selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces delbrückii, Saccharomyces italicus, Saccharomyces ellipsoideus, Saccharomyces fermentati, Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis, Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomyces montanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis, Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum and Saccharomycodes ludwigii , as well as yeasts of the species Kluyveromyces such as K.
  • yeasts of the species Candida such as Candida utilis, Candida tropicalis, Candida albicans, Candida lipolytica and Candida versatilis , and yeasts of the species Pichia such as Pichia stipidis, Pichia pastoris and Pichia sorbitophila , and yeasts of the species Cryptococcus, Debaromyces, Hansenula, Saccharomycecopsis, Saccharomycodes, Schizosaccharomyces, Wickerhamia, Debayomyces, Hanseniaspora, Kloeckera, Zygosaccharomyces, Ogataea, Kuraishia, Komagataella, Metschnikowia, Williopsis, Nakazawaea, Cryptococcus, Torulaspora, Bullera, Rhodotorula, Willopsis and Sporobolomyces , and mosses such as Physcom
  • the above organism is the basis or a tool for generating an additionally genetically modified organism, which functionally heterologously expresses a mammal membrane protein. Therefore, it is furthermore preferred that the organism additionally is transformed with a gene coding for a membrane protein of a mammal, in particular a human membrane protein, preferably a plasma membrane protein, under the control of a preferably constitutively active promoter.
  • the gene coding for the membrane protein is in principle arbitrary and preferably is selected from the group or family consisting of serotonin transporter gene, noradrenaline transporter gene, dopamine transporter gene, choline transporter gene, glycine transporter gene, gamma-amino acid transporter gene, 5-hydroxytryptamine receptor 3 subunit C, mast/stem cell growth factor receptor precursor, toll-like receptor 8 precursor, similar to olfactory receptor MOR233-18, green-sensitive opsin, glycine receptor, frizzled 5 precursor, dimethylaniline monooxygenase [N-oxide forming], calcitonin gene-related peptide type 1 receptor precursor, muscarinic acetylcholine receptor, C—C chemokine receptor type 4, G protein-coupled receptor, receptor tyrosine kinase, dopamine receptor, substance-K receptor, putative neurotransmitter receptor, thyroid hormone receptor-associated protein complex component TRAP230, fMet-Leu
  • HMG-CoA reductases including truncated, but still functional HMG-CoA reductases such as tHMG1, are known for instance from the following Acc.-Nos. of the gene database of The National Center for Biotechnology Information (NCBI): NC — 001145, NM — 106299, NC — 003421.2, NC — 009784.1, NC — 003028.3, NC — 007-308.3, and FIG. 3 (tHMG1).
  • NCBI National Center for Biotechnology Information
  • NCBI National Center for Biotechnology Information
  • NM — 103926 Gene sequences coding for suitable delta7 reductases are known for instance from the following Acc.-Nos. of the gene database of The National Center for Biotechnology Information (NCBI): NM — 103926, NM — 001360, NM — 007856, NM — 203904, NM — 001014927, NM — 201330, NM — 022389, NM — 001131727, NM — 001087087, XM — 001497598, XM — 001174160, XM — 001-099101, BM490402, CA753545.
  • NCBI National Center for Biotechnology Information
  • NM — 014762 NM — 001016800, NM — 001094456, NM — 001-008645, NM — 001103276, NM — 001080148, NM — 053272, NM — 00103128, XM — 001488247, AB125202, XM — 001153751.
  • NCBI National Center for Biotechnology Information
  • NCBI National Center for Biotechnology Information
  • Gene sequences coding for exemplary heterologously expressible mammal membrane proteins are known for instance from the following Acc.-Nos. of the gene database of The National Center for Biotechnology Information (NCBI): NP — 570126, NP — 006-019, NP — 000860, NP — 619542, NP — 000786, NP — 000787, NP — 150645, NP — 150646, NP — 057658, NP — 387512, NP — 00-0669, NP — 150647, NP — 000671, NP — 387507, NP — 387508, NP — 387509, NP — 000670, NP — 001690, NP — 068713. Further gene sequences for mammal membrane proteins, in particular for the membrane proteins listed above, can easily be identified by research in the mentioned gene database.
  • promoters of the respective heterologously expressed enzymes and/or proteins may be respectively identical or different in an organism according to the invention
  • suitable promoters are known for instance from the following Acc.-Nos. of the gene database of The National Center for Biotechnology Information (NCBI): NC — 001142, NC — 001139, NC — 001147, NC — 001139, NC — 001148, NC — 001135, NC — 001136.
  • An enzymatic activity in an organism according to the invention is increased, compared to the wild type, if the activity is at least by 10%, preferably at least by 50%, most preferably at least by 1,000% higher than the same activity in the corresponding wild type.
  • the term increase also comprises the presence of the respective enzymatic activity in an organism according to the invention, if the corresponding wild type does not have any identical activity.
  • An enzyme has HMG-CoA reductase activity, if it is capable of catalyzing the reaction of beta-hydroxy-beta-methylglutaryl coenzyme A to mevalonate.
  • An enzyme has C24 methyltransferase activity, if it is capable of catalyzing the reaction of zymosterol to fecosterol.
  • An enzyme has delta22 desaturase activity, if it is capable of catalyzing the reaction of ergosta-5,7,24(28)-trienol to ergosta-5,7,22,24(28)-tetraenol.
  • An enzyme has dehydrocholesterol-delta7 reductase (in short: delta7 reductase) activity, if it is capable of catalyzing the reaction of cholesta-5,7,24-trien-3-ol to desmosterol.
  • An enzyme has dehydrocholesterol-delta24 reductase (in short: delta24 reductase) activity, if it is capable of catalyzing the reaction of cholesta-7-24-dien-3beta-ol to lathosterol, of cholesta-5,7,24-trien-3-ol to 7-dehydrocholesterol, and/or of desmosterol to cholesterol.
  • An enzyme has squalene epoxidase activity, if it is capable of catalyzing the reaction of squalene to squalene epoxide.
  • An enzyme has lanosterol demethylase activity, if it is capable of catalyzing the reaction of lanosterol to 4,4-dimethylcholesta-8,14,24-trienol.
  • An enzyme has delta7-delta8 isomerase activity, if it is capable of catalyzing the reaction of fecosterol to episterol, and/or of zymosterol to cholesta-7-24-dien-3-beta-ol.
  • Such an enzyme is preferably expressed in the wild type of the organism according to the invention. If the wild type of this enzyme is not or not to a sufficient degree expressed, the organism according to the invention can be transformed with a gene coding for this enzyme, under the control of a preferably constitutively active promoter.
  • An enzyme has delta5 desaturase activity, if it is capable of catalyzing the reaction of episterol to ergosta-5-7-24(28), of cholesta-7-24-N dien-3-beta-ol to cholesta-5,7,24-trien-3-ol, and/or of lathosterol to 7-dehydrocholesterol.
  • Such an enzyme is preferably expressed in the wild type of the organism according to the invention already. If the wild type of this enzyme is not or not to a sufficient degree expressed, the organism according to the invention can be transformed with a gene coding for this enzyme, under the control of a preferably constitutively active promoter.
  • an enzymatic activity mentioned above is measured by that a given amount of the respective educt is reacted to the product under addition of a given amount of the respective enzyme and if applicable given amounts of the other necessary reaction partners, and the amount of the product formed in a given time is determined.
  • the determination of the HMG-CoA reductase activity, the lanosterol demethylase activity, the squalene epoxidase activity, the C24 methyltransferase activity, the delta7-delta8 isomerase activity, the delta5 desaturase activity, the delta desaturase activity and the delta24 reductase activity is made for instance as described in the document WO 03/064650 A1.
  • the determination of the delta7 reductase activity is made in analogous manner.
  • the content of desmosterol and/or cholesterol is increased in a membrane, in particular a plasma membrane, of an organism according to the invention compared to the wild type, if the amount of the respective substance is at least by 10%, preferably at least by 50%, most preferably at least by 1,000% higher than the same activity in the corresponding wild type.
  • the term increase also comprises the presence of the respective substance in the membrane of an organism according to the invention, if the substance cannot be detected in the membrane of a corresponding wild type.
  • the measurement of the amount is performed as mentioned in the examples under the headings “Processing of the membrane sterols” and “Conditions for the gas chromatography (GC)”.
  • the invention further teaches the use of an organism according to the invention for the production of desmosterol and/or cholesterol, the organism preferably being aerobically cultivated.
  • the suitable cultivation conditions for a certain organism are well known to a person skilled in the art.
  • an organism according to the invention teaches the production of a membrane protein of a mammal, in particular of a human membrane protein, preferably of a plasma membrane protein, and is characterized in that the organism transformed with a gene coding for a membrane protein of a mammal is cultivated, preferably without addition of cholesterol and/or desmosterol, and that after expiration of a given cultivation time the membrane protein is isolated from a cultivation supernatant and/or from the organism.
  • an organism according to the invention relates to the screening for substances binding to a membrane protein of a mammal, in particular to a human membrane protein, preferably to a plasma membrane protein, wherein the organism transformed with the gene for the membrane protein or a membrane extract of the transformed organism, if applicable after previous cultivation of the organism, is contacted with a given substance or a mixture of given substances, that modifications or absence of modifications of properties of the organism, of the membrane extract, and/or the binding or non-binding of a substance to the membrane protein is measured, and that with measurement of modifications and/or binding, the substance or the mixture of substances is categorized as binding to the membrane protein.
  • the binding of a prospective substance to a membrane protein can be made with all conventional binding assays.
  • the membrane protein and/or the substance is covalently bound to a reporter molecule detectable by means of physical, physicochemical or chemical methods.
  • the invention further relates to a kit for carrying-out a screening method according to the invention.
  • It contains an organism according to the invention, optionally a nucleic acid construct suitable for the transformation of the organism and containing a nucleic acid coding for a membrane protein of a mammal, in particular a human membrane protein, preferably a plasma membrane protein, which is under the control of a preferably constitutively active promoter, and instructions of use for transforming the organism with the nucleic acid construct contained or separately made available, for cultivating the organism before and after the transformation, for contacting the organism with a given substance or a given mixture of substances, and for measuring a modification of a property of the organism and/or a binding of the substance or mixture of substances to the membrane protein.
  • nucleic acid construct in particular plasmid or shuttle vector, containing a nucleic acid or several nucleic acids, identical or different, coding for a protein with dehydrocholesterol-delta7 reductase activity and/or dehydrocholesterol-delta24 reductase activity, wherein the nucleic acid(s) is or are (respectively) under the control of a preferably constitutively active promoter.
  • the nucleic acid construct may additionally contain: a nucleic acid or several nucleic acids, identical or different, coding for a protein with HMG-CoA reductase and/or squalene epoxidase and/or lanosterol demethylase activity, wherein the nucleic acid(s) is or are (respectively) under the control of a preferably constitutively active promoter.
  • nucleic acid construct can be used for producing an organism according to the invention, wherein a precursor organism, with which the nucleic acid construct is transformed, wherein the precursor organism preferably is a genetically modified organism having a reduced C24 methyltransferase and/or a delta22 desaturase activity compared to the wild type.
  • the invention relates to a membrane extract, in particular plasma membrane extract, obtainable from an organism according to the invention by digestion of the organism and separation of the membrane, in particular the plasma membrane. Suitable methods for the digestion or for the separation and isolation of the membrane are well known to the man skilled in the art.
  • the eukaryotic expression vector pCis contains the cDNA of the rat serotonin transporter, which was integrated between the restriction sites XhoI and HpaI.
  • yeast extract 0.5% yeast extract; 2% glucose; pH 6.3.
  • YNB yeast nitrogen base
  • Ampicillin (Boehringer, Mannheim) 50 ⁇ g/ml.
  • Leucine (0.4 g/l); histidine-HCl (20 mg/l).
  • PBS Phosphate-Buffered Saline
  • E. coli was performed in 100 ml conical flasks at 37° C. and 160 rpm on a rotary shaker in LB medium.
  • the antibiotic ampicillin 50 ⁇ g/ml
  • Precultures of the yeast S. cerevisiae were prepared at 30° C. and 160 rpm in 100 ml conical flasks and main cultures were prepared in 250 ml baffled flasks on a rotary shaker. If not explicitly described otherwise, all cultivations of the yeast S. cerevisiae were performed in WMVIII medium (C. Lang, A. C. Looman. Appl. Microbiol. Biotechnol. (1995) 44: 147-156), supplemented with 1 mg/ml histidine.
  • the cultivation of the strain S. cerevisiae GRFura3 pFlat3-rSERT or pFlat3-leer was made under strictly anaerobic conditions, as follows: A 20 ml preculture was prepared, as mentioned under “Aerobic cultivation”, for 48 h. The main culture was inoculated with 1% with this preculture and cultivated in a 250 ml baffled flask in 50 ml WMVIII medium, supplemented with 1 mg/l histidine and 40 mg/l cholesterol, under strictly anaerobic conditions at 30° C. and 160 rpm on a rotary shaker.
  • the baffled flask was incubated in a 2.5 l anaerobic jar (Merck, Darmstadt) together with two gas packs (Anaerocult A, Merck, Darmstadt).
  • the cholesterol supplementation was made with 0.5 ml (pro 50 ml medium) of a cholesterol solution (2.5 g ethanol; 2.5 g Tween 80; 20 mg cholesterol), so that a final concentration of 40 mg cholesterol per 1 liter medium resulted.
  • the restriction of the DNA (1 to 10 ⁇ g) was made in 30 ⁇ l batches, consisting of DNA, 3 ⁇ l of the corresponding buffer, 1 ⁇ l RSA (3 mg/ml) and 1 U enzymes (NotI and XhoI, BioLabs, Schwalbach) per 1 ⁇ g used DNA.
  • the batch was incubated at 37° C. for two hours.
  • an incubation for four hours was made.
  • the agarose gel electrophoresis was made in mini-gel apparatuses (BioRad, Kunststoff). The gels consisted of 1% agarose in TAE buffer. As electrophoresis buffer, TAE buffer was again used. 10 ⁇ l of the sample were reacted with 3 ⁇ l of a stopper solution. For fragments between 2 kb and 13 kb, ADNA (BioLabs, Schwalbach) restricted with HindIII (band size: 23.1 kb; 9.4 kb; 6.6 kb; 4.4 kb; 2.3 kb; 2.0 kb and 0.6 kb) served as standard. For smaller fragments between 500 bp and 3 kb, the marker GeneRuler (MBI Fermentas, St.
  • Leon-Rot was used, and for fragments between 3 kb and 10 kb, the marker 1 kb DNA Ladder (New England Biolabs) was used. For the fractionation of DNA fragments, a voltage of 100 V was applied for 30-45 min. Then, the gel was dyed in an ethidium bromide solution (0.4 mg/l TAE buffer) and received under UV light (254 nm).
  • DNA from aqueous solutions was concentrated by means of a precipitation.
  • the DNA solution was reacted with 1/10 volume 3 M sodium acetate (pH 4.8) and a 2.5 times larger volume ethanol (96%).
  • the batch was incubated for 30 min at ⁇ 20° C. and then centrifuged at 14,000 g.
  • the DNA pellet was then again washed with 70% ethanol and centrifuged at 14,000 g for 5 min.
  • the air-dried pellet was resuspended in a desired volume of TE buffer and stored at ⁇ 20° C.
  • the T4-DNA ligase catalyzes the linking of co-valent phosphodiester bonds between the 3′-hydroxyl group of a nucleotide and the 5′-phosphate group of a second nucleotide in a double-strand DNA.
  • the fragments to be ligated were purified by means of a DNA purification kit (Qiagen, Hilden) following the manufacturer's instructions and unified in a vector to insert DNA proportion of 3:1 and received in a volume of 17 ⁇ l. Thereafter, 2 ⁇ l ligase buffer (MBI Fermentas, St. Leon-Rot) and 1 ⁇ l T4 ligase (1 U/ ⁇ l) were added and the batch was incubated for 2 hours in the case of a “sticky end ligation” at 16° C.
  • E. coli cells with plasmid DNA were made using the electroporation method.
  • electrocompetent cells an over-night culture was inoculated by 1% in 500 ml fresh LB medium and aerobically grown at 37° C. up to an optical density of 0.5-0.7. Then, the cells were centrifuged at 5,000 g for 10 min and resuspended in 200 ml ice-cold 10% glycerol. After another centrifugation step at 5,000 g for 10 min, the cells were resuspended in 100 ml ice-cold 10% glycerol.
  • the batch was transferred into sterile electroporation cuvettes pre-cooled on ice, and the electroporation (1.67 kV, 25 ⁇ F, 200 ⁇ , 5 ms) was performed with a Multiporator (Eppendorf, Hamburg). After pulse application, the immediate addition of 1 ml YE medium and the transfer into a 1.5 ml Eppendorf vessel took place. The batch was now incubated for regeneration for 1 h at 37° C. and then plated-out on LB-agar plates with ampicillin for selection.
  • the respective E. coli strains were inoculated in 5 ml LB medium with 50 mg/l ampicillin and grown over night at 37° C. The cells were then centrifuged at 5,000 g. The supernatant was removed and the plasmid DNA was processed by means of a Miniprep kit (Qiagen, Hilden) following manufacturer's instructions.
  • yeast transformation For the yeast transformation, a preculture in 20 ml YE medium was cultivated over night. A 50 ml main culture was inoculated to an optical density OD 600 of 0.3 and cultivated for approx. 4 hours. When an OD 600 of 0.8-1.0 was obtained, the cells were centrifuged at 4,000 g for 10 min, and the cells pellet was washed with 10 ml sterile H 2 O. Thereafter, the cells were received in 1.5 ml H 2 O and transferred into an Eppendorf vessel.
  • the cells were centrifuged (4,000 g, 2 min) and resuspended in 1 ml lithium acetate solution (100 mM LiAc in TE buffer), centrifuged (4,000 g, 2 min) and again washed with 1 ml lithium acetate solution. Then, the cells were received in 200 ⁇ l lithium acetate solution (competent cells).
  • competent cells 10 ⁇ l herring sperm DNA (10 mg/ml), 230 ⁇ l PEG solution (40% PEG 4000 in 0.1 M lithium acetate in 1 ⁇ TE pH 8.0) and 1-20 ⁇ g DNA to be transformed were used. This batch was incubated for 30 min at 30° C.
  • the transformation batch contained approx. 30 ⁇ g of each vector.
  • the vector DNA was precipitated and received in 10 ⁇ l H 2 O.
  • 40 ⁇ l competent cells were added and this batch was incubated for 30 min at 37° C., before the PEG solution and the herring sperm DNA were added. Thereafter as described above.
  • yeast cultures were carried out according to the following scheme: A 20 ml preculture was inoculated with 50 ⁇ l of a frozen culture and cultivated for 2 days. The main culture was carried out in 250 ml baffled flasks with 50 ml medium (WMVII I medium, supplemented with 1 mg/ml histidine) on a rotary shaker at 160 rpm and 30° C. for 1 or 3 days, respectively. The main culture was inoculated with 1% of the preculture.
  • WMVII I medium 50 ml medium
  • the main culture was inoculated with 1% of the preculture.
  • the cells to be investigated were harvested and washed with 40 ml H 2 O. Then, the optical density OD 600 was determined and a yeast pellet, corresponding to 125 OD 600 was received in 5 ml 0.5 N HCl solution. The suspension was then boiled in a 50 ml Falcon tube for 20 min at 100° C. The thus labilized cells were thereafter cooled down on ice and 3 g KOH were added to the solution. After dissolving of the KOH, 12.5 ml of a 0.25 g/1 methanolic pyrogallol solution were added. As an extraction standard, 10 ⁇ l cholesterol solution (10 mg/ml) were additionally added. The saponification mixture was incubated for 1 h 45 min at 70° C.
  • the sterols were extracted with 2 ⁇ 20 ml n-hexane.
  • the combined n-hexane phases were concentrated at the rotary evaporator and the residue was dissolved with 2 ⁇ 1 ml chloroform with 0.2 g/l nonadecane as an internal standard and transferred into brown glass bottles.
  • the sterol extracts were stored at ⁇ 20° C. before analysis.
  • the purified plasma membrane could be removed with a Pasteur pipette from the interphase and was transferred into a new Beckman tube, which was filled up with H 2 O.
  • the plasma membrane was thereafter pelletized at 80,000 g for 30 min and the pellet was resuspended in 2 ml TED buffer and stored at ⁇ 20° C.
  • 1 ml of the plasma membrane suspension was reacted with 500 ⁇ l chloro-form and vortexed for 1 min. Then followed the phase separation at 13,000 rpm for 15 min.
  • the GC analysis was carried out with an Agilent 6890N gas chromatograph (Agilent, Waldbronn), equipped with an Autosampler Agilent 7683B. As a detector, an Agilent 5975 VL mass spectrometer was used. The following conditions were selected: The column was a 30 m long HP-5MS column (J&W Scientific, USA) with an inner diameter of 0.25 mm and a film thickness of 0.25 ⁇ m. Helium served as mobile phase. The GC/MS system was operated with a temperature program (100° C. 0.5 min, 50° C./min to 270° C., 270° C. for 10 min, 5° C./min to 290° C., 290° C.
  • the injector temperature was 280° C., that of the detector was 150° C.
  • the sterol sample were derivatized with MSTFA for one hour at 80° C. in the heating oven.
  • the injection volume of the samples was 2 ⁇ l.
  • the gene URA3 was deleted and the gene tHMG1 was integrated at the gene locus LEU2.
  • the transcription of the gene tHMG1 is controlled by the constitutive ADH1 promoter.
  • ERG1 and ERG11 were integrated in the yeast genome in a transcriptionally deregulated form under the control of a constitutive ADH1 promoter, and that by means of homologous recombination at the loci of the genes ERG5 and ERG6, which thereby were deleted (Veen, 2002, see above).
  • a dehydrocholesterol-delta7 reductase (known from the document U.S. Pat. No. 5,759,801) was brought to overexpression in this strain by means of the shuttle vector pFlat1.
  • DHCR7 dehydrocholesterol-delta7 reductase
  • Table 2 shows the composition of the main sterols in the yeast GRFcol after transformation with the vector pFLAT1-DHCR7, which contains the dehydrocholesterol- ⁇ 7 reductase from Arabidopsis thaliana , and/or pFlat3-DHCR24, which contains the dehydrocholesterol-A24 reductase from Xenopus laevis .
  • the values originate from a GC/MS analysis and are percentage shares.
  • a dehydrocholesterol- ⁇ 24 reductase was expressed in the yeast additionally to DHCR7.
  • the cDNA for the dehydrocholesterol-A24 reductase (DHCR24) from Xenupus laevis was integrated between the constitutive ADH1 promoter and the tryptophan terminator (TRP1) of the pFlat3 vector and introduced into the yeast.
  • the resulting strain was designated GRFcol pFlat1-DHCR7 pFlat3-DHCR24.
  • the serotonin transporter of the rat was overexpressed in the strain GRFcol pFlat1-DHCR7.
  • the rSERT-cDNA was integrated between the constitutive ADH1 promoter (C. Lang, A. C. Looman, Appl. Microbiol. Biotechnol. 44: 147-156 (1995)) and the tryptophan terminator (TRP1) of the pFlat3 vector.
  • TRP1 tryptophan terminator
  • the pFlat vector system is diagrammatically shown in FIG. 1 . It shows a diagrammatic representation of the episomal yeast expression plasmids pFlat1 and pFlat3.
  • the plasmid pFlat1 carries a URA3 gene and pFlat3 carries a LEU2 gene for the selection of plasmid-carrying strains.
  • Both plasmids contain the constitutive ADH1 promoter and the TRP1 terminator, as well as the largest part of the 2 ⁇ m DNA for the replication.
  • the cloning of the rSERT cDNA between the restriction sites NotI and XhoI of the multiple cloning site is diagrammatically shown.
  • the strain GRFcol pFlat1-DHCR7 as well as the wild type strain GRF18 were transformed with the plasmid, and the serotonin receptor was brought to expression.
  • the functional expression of the rSERT in the yeast strains to be investigated was verified by binding [ 3 H] citalopram to spheroblasts.
  • Serotonin antagonists, such as citalopram exclusively bind to correctly folded and active, i.e. to functionally expressed SERT molecules.
  • Citalopram binds with high affinity to the same site of the serotonin transporter as the actual substrate, serotonin.
  • citalopram reduces the maximum number of the binding sites, does however not lower the affinity of the serotonin for the SERT. It is known that [ 3 H] citalopram only binds to the rSERT, if cholesterol is present in the surrounding membrane. Ergosterol could not replace cholesterol in this study. Whether this is possible with the sterols cholesta-5,7,24-trienol and desmosterol, which are more similar to the structure of cholesterol, should be investigated in an experiment.
  • FIG. 2 shows the results of this study. Shown is a scintillation count of the bound [ 3 H] citalopram in fmol per 10 9 cells.
  • FIG. 2 shows that the non-specific binding is highest for GRFcol-DHCR7 and lowest for GRFura3 anaerobic+cholesterol.
  • FIG. 1 A first figure.

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