Microorganism for expressing a human membrane protein

Abstract

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 reduced C24-methyltransferase and/or delta22-desaturase activity compared to the wild type. The invention is characterized in that the organism has increased dehydrocholesterol-delta70-reductase activity compared to the wild type. The invention further relates to different uses of such an organism, to a test kit comprising such an organism, and to a membrane extract of such an organism.

1 . 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. 
     
     
         2 . The organism of  claim 1 , wherein the organism additionally has an increased dehydrocholesterol-delta24 reductase activity compared to the wild type. 
     
     
         3 . The organism of  claim 1 , wherein the organism has an increased squalene epoxidase and/or lanosterol demethylase activity compared to the wild type. 
     
     
         4 . The organism of  claim 1 , wherein the organism is a prokaryotic organism, preferably selected from the group consisting of bacteria of the species  Bacillus, Escherichia, Lactobacillus, Corynebacterium, Acetobacter, Acinetobacter  and  Pseudomonas  or a eukaryotic organism, most preferably selected from the group consisting of algae, yeasts, fungi, insect cells, mosses and plants. 
     
     
         5 . The organism of  claim 4 , wherein the yeast is selected from the group consisting of  Saccharomyces cerevisiae, Saccharomyces delbrückii, Saccharomyces italicus, Saccharomyces ellipsoideus, Saccharomyces fermen - tati, Saccharomyces kluyveri, Saccharomyces kru - sei, Saccharomyces lactis, Saccharomyces marx - ianus, Saccharomyces microellipsoides, Saccharomyces montanus, Saccharomyces norbensis, Saccharomy - ces 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. lactis K. marxianus  var.  marxianus, K. thermotol - erans , and yeasts of the species  Candida  such as  Candida utilis, Candida tropicalis, Candida albi - cans, 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, Debaromy - ces, Hansenula, Saccharomycecopsis, Saccharomy - codes, Schizosaccharomyces, Wickerhamia, Debayomy - ces, Hanseniaspora, Kloeckera, Zygosaccharomyces, Ogataea, Kuraishia, Komagataella, Metschnikowia, Williopsis, Nakazawaea, Cryptococcus, Torulaspora, Bullera, Rhodotorula, Willopsis  and  Sporobolomyces.    
     
     
         6 . The organism of  claim 1 , comprising, preferably a membrane, most preferably a plasma membrane, with an increased content of desmosterol and/or cholesterol and/or 7-dehydro-cholesterol and/or lathosterol compared to the wild type. 
     
     
         7 . The organism of  claim 1 , wherein the organism is additionally transformed with a gene coding for a membrane protein of a mammal, preferably a human membrane protein, most preferably a plasma membrane protein, under the control of a preferably constitutively active promoter. 
     
     
         8 . A method of using an organism of  claim 1  comprising aerobically cultivating the organism for the production of desmosterol and/or cholesterol and/or 7-dehydrocholesterol. 
     
     
         9 . The method of  claim 8  further comprising cultivating the transformed organism without addition of cholesterol and/or desmosterol, and isolating the membrane protein after expiration of a given cultivation time from a cultivation supernatant and/or from the organism for the production of a membrane protein of a mammal, preferably of a human membrane protein, most preferably of a plasma membrane protein. 
     
     
         10 . The method of  claim 8  further comprising contacting the organism or a membrane extract of the organism, after previous cultivation of the organism, with a given substance or a mixture of given substances; measuring the 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; and categorizing with measurement of modifications and/or binding the substance or the mixture of substances as binding to the membrane protein for screening for substances that bind to a membrane protein of a mammal, preferably to a human membrane protein, most preferably to a plasma membrane protein. 
     
     
         11 . A kit comprising an organism of  claim 1 , optionally with a nucleic acid construct suitable for the transformation of the organism and containing a nucleic acid coding for a membrane protein of a mammal, preferably a human membrane protein, most preferably a plasma membrane protein, which is under the control of a preferably constitutively active promoter, and comprising instructions of use for transforming the organism with the nucleic acid construct, for cultivating the organism before and after the transformation, contacting the organism with a given substance or a given mixture of substances, and measuring a modification of a property of the organism and/or a binding of the substance or mixture of substances to the membrane protein. 
     
     
         12 . A nucleic acid construct, preferably a plasmid or shuttle vector, comprising 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. 
     
     
         13 . The nucleic acid construct of  claim 12 , further comprising 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. 
     
     
         14 . A method of using a nucleic acid construct of  claim 12  comprising transforming a precursor organism with the nucleic acid construct, wherein the precursor organism preferably is a genetically modified organism having a reduced C24 methyltransferase and/or delta22 desaturase activity compared to the wild type for producing an isolated genetically modified living non-mammal organism. 
     
     
         15 . A membrane extract, preferably a plasma membrane extract, obtainable from an organism of  claim 1  by digestion of the organism and separation of the membrane, preferably of the plasma membrane.