Compositions and method for stable isotope labelling of biological compounds
First Claim
1. A method for producing a nutrient medium for growing mammalian or insect cells in culture whereby for at least one of H, C or N, substantially all atoms in substrates that are used by the cells for synthesis of biomolecules in the nutrient medium are isotopically labelled, whereby the method comprising the steps of:
- (a) growing an organism on a mineral medium which supports growth of the organism, whereby in the medium substantially all of the assimilable atoms, for at least one of H, C or N, are isotopically labelled, to produce labelled biomass;
(b) autolysing the biomass of an organism grown as in (a) to produce an autolysate; and
, (c) composing the nutrient medium by combining the autolysate as obtained in (b) with further components necessary for growth of the mammalian or insect cells.
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Abstract
The present invention is concerned the labelling of biological compounds with stable isotopes such that the three-dimensional structure of the biological compounds may be analysed by e.g. NMR spectroscopy. The invention employs microorganisms that are grown on mineral media comprising carbon and nitrogen sources that contain stable isotopes to produce biomass that is uniformly labelled with stable isotopes. The biomass may be autolysed to produce an autolysate. The biomass may further be extracted with organic solvent to produce lipids. The (delipidised) biomass is hydrolysed to produce labelled amino acids and other nutrients, which are used together with the autolysate, extracted lipids and further components to compose a culture medium for a mammalian or insect host cells for the production of biological compounds that are uniformly labelled with stable isotopes. The biological compound preferably is a biological acromolecule, such as e.g. a mammalian membrane protein. Data supplied from the esp@cenet database—Worldwide
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Citations
30 Claims
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1. A method for producing a nutrient medium for growing mammalian or insect cells in culture whereby for at least one of H, C or N, substantially all atoms in substrates that are used by the cells for synthesis of biomolecules in the nutrient medium are isotopically labelled, whereby the method comprising the steps of:
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(a) growing an organism on a mineral medium which supports growth of the organism, whereby in the medium substantially all of the assimilable atoms, for at least one of H, C or N, are isotopically labelled, to produce labelled biomass;
(b) autolysing the biomass of an organism grown as in (a) to produce an autolysate; and
,(c) composing the nutrient medium by combining the autolysate as obtained in (b) with further components necessary for growth of the mammalian or insect cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for obtaining structural information on a biomolecule, the method comprising the steps of:
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(a) producing a biomolecule, whereby substantially all atoms in the biomolecule are isotopically labelled, in a method according to;
(b) optionally, purifying the biomolecule;
(c) subjecting the biomolecule to spectroscopic analysis to obtain information about its structure. - View Dependent Claims (18, 19, 20, 21, 22)
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23. A nutrient medium for the production of an isotopically labelled biomolecule from mammalian or insect cells, the medium supporting growth of a mammalian or insect cell culture under condition conducive to the production of the biomolecule, the medium comprising:
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(a) a mixture of inorganic salts;
(b) a source of amino acids;
(c) a carbohydrate energy source;
(d) a source of lipids;
(e) optionally, a protective agent;
(f) optionally, vitamins and/or organic compounds;
(g) optionally, organic acids; and
, (h) optionally, trace elements;
whereby substantially all atoms in (a), (b), and (c), and, optionally in (d), (e), (f), (g) and (h) are isotopically labelled for at least one of H, C or N or whereby 20-100% of the hydrogen atoms in (a), (b), and (c), and, optionally in (d), (e), (f), (g) and (h) are uniformly substituted with the isotope 2H. - View Dependent Claims (24, 25, 26, 27)
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28. A mammalian membrane protein whereby substantially all atoms in the protein are isotopically labelled with an isotope selected from 15N, 13C 2H 15N and 13C, ′
- 5N and 2H, 13C, and 2H, or 13C and 2H.
- View Dependent Claims (30)
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29. A mammalian membrane protein whereby 20-100% of the hydrogen atoms in the protein are uniformly substituted with the isotope 2H.
Specification