Microorganism for expressing a human membrane protein

a technology of membrane protein and microorganism, which is applied in the field of isolated genetically modified living non-mammal organisms, can solve the problems of heterologous expression of membrane protein from mammal cells in wild type strains, difficulty in expressing sert,

Inactive Publication Date: 2012-06-21
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]It is therefore the technical object of the invention to provide a non-mammal organism, in particular a microorganism, in which mammal membrane proteins, in particular human membrane proteins, can be functionally expressed in an efficient and simple way.
[0016]It is another technical object of the invention to provide a means, which permits in a simple and efficient way the screening of substances or substance libraries for agonists or antagonists of the mammal membrane proteins, in particular the human membrane proteins.

Problems solved by technology

The heterologous expression of membrane proteins from mammal cells in wild type strains, however, 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.
The SERT is an extremely difficult protein with regard to heterologous or overexpression.
Difficulties occur mainly by the dependence of the serotonin transporter on cholesterol in the membrane surrounding the protein and the importance of the glycosylation for the correct folding of the protein.

Method used

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  • Microorganism for expressing a human membrane protein
  • Microorganism for expressing a human membrane protein
  • Microorganism for expressing a human membrane protein

Examples

Experimental program
Comparison scheme
Effect test

example 1

Used Materials and Methods

1.1: Primers.

[0058]All primers were synthesized by the company Metabion (Martinsried).

TABLE 1PCR primers for amplification. The recognitionsequences for the restriction enzymes NotIand XhoI are underlined.PrimerSequencemSERTNotIf5′-ATGCGGCCGCACCATGGAGACCACACCTTTG-3′mSERTXhoIr5′-ATCTCGAGTTACACAGCATTCATGCGG-3′

1.2: Plasmids.

[0059]pFlat vector system (Veen, M., dissertation, TU Berlin (2002)).

[0060]In the following experiments, different strains of the yeast S. cerevisiae were transformed with the pFlat1 or pFlat3 vector of the pFlat vector system (Jacobs B L, Azmitia E C. Physiol Rev (1992) 72, 165-229). These E. coliS. cerevisiae “shuttle vectors” comprise a NotI and a XhoI restriction site in the “Multiple cloning site”, which were used in the present work for cloning. Furthermore, these plasmids carry the LEU2 (pFlat3) or the URA3 gene (pFlat1), respectively, which are used as auxotrophy marker genes for the selection of plasmid-carrying strains on correspo...

example 2

Results

[0092]In the year 2002, the biosynthesis of cholesta-5,7,24-trien-3-ol in the yeast S. cerevisiae could be established (Veen, 2002, see above). For this purpose, a freely obtainable lab strain GRF18 as shown in the following was modified with genetic methods.

StrainDescriptionGRFl8↓leu2, his3GRFura3↓leu2, his3, ura3GRFtH3↓tHMG1-leu2, his3, ura3GRFtH3E1e5↓tHMG1-leu2, ERG1-erg5, his3, ura3GRFtH1E1E11e5e6tHMG1-leu2, ERG1-erg5, ERG11-erg6,his3, ura3

[0093]As can be seen, first the gene URA3 was deleted and the gene tHMG1 was integrated at the gene locus LEU2. The gene tHMG1 (t=truncated, cut short) codes for a cut short HMG-CoA reductase, which only consists of the catalytic subunit of the protein and is thus not subject anymore to the feedback inhibition by sterol intermediates. The transcription of the gene tHMG1 is controlled by the constitutive ADH1 promoter. By the analysis of a systematic overexpression and transcriptional deregulation of all genes of the post-squalene biosyn...

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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.

Description

FIELD OF THE INVENTION[0001]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.PRIOR ART AND BACKGROUND OF THE INVENTION[0002]An organism of the structure mentioned above is known from the document WO 03 / 064650. This organism is suitable for the biosynthesis of 7-dehydrocholesterol. The motivation for creating this organism is based on the need to produce precursor products to vitamin D3, in particular 7-dehydrocholesterol, in large amounts and under economical conditions.[0003]In a completely different context, there is a need of membrane proteins, in particular human membrane proteins, functionally in a native conformation in at...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P33/00C12N1/13G01N33/53C12N1/15C12N5/10C12N15/63C12N1/21C12N1/19
CPCC07K14/705C12N15/81C12P33/00C12P21/02
Inventor SCHILLING, MICHAELLANG, CHRISTINERAAB, ANDREAS
Owner NOVOZYMES AS
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