Compositions and method for stable isotope labelling of biological compounds

• US 20070082399A1
• Filed: 07/12/2004
• Published: 04/12/2007
• Est. Priority Date: 07/11/2003
• Status: Abandoned Application

• Current Assignee: Tatiana A. Egorova-Zachernyuk
• Original Assignee: Tatiana A. Egorova-Zachernyuk
• Inventors: Egorova-Zachernyuk, Tatiana
• Application Number:
  US10/564,012
• Publication Number:
  US 20070082399A1
• Time in Patent Office: Days
• US Class Current: 435/404
• CPC Class Codes: C12N 1/06 Lysis of microorganisms
• CPC Class Codes: C12P 13/04 Alpha- or beta- amino acids other amino acids C12P13/005
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

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

Claims

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.

2. A method according to claim 1, wherein the organism is a fungus, yeast or algae.

3. A method according to claim 2, wherein the organism is an organism that belongs to a genus selected from Saccharontyces, Pichia, Hansenula, Kluyveroznyces, Candida, Brettanonzyces, Debaryonzyces, Tolrulopsis, Yarrowia, Galdieria, Cyanidiunt, Porphyridiunz, Cystocloniunz, Audouinella, and Cyanidioschyzon.

4. A method according to claim 1, wherein the method further comprises 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) extracting biomass of an organism with an organic solvent to produce an extract comprising lipids, whereby the organism is grown as in (a) or is grown as in (a) on a medium without isotopic substitution;

(c) hydrolysing biomass of an organism grown as in (a) at a non-alkaline pH to produce a hydrolysate comprising amino acids;

(d) composing the nutrient medium by combining the autolysate as obtained in any one of claims 1-3 with amino acids as obtained in (c) and adding further components necessary for growth of the mammalian or insect cells.

5. A methods according to claim 4, wherein in step (d) the nutrient medium is composed by combining the autolysate with the amino acids and the lipids and adding further components necessary for growth of the mammalian or insect cells.

6. A method according to claim 1, whereby the nutrient medium is composed of autolysate, lipids and amino acids obtained from at least two different organisms.

7. A method according to claim 1, whereby, prior to hydrolysis in (c), lipids and pigments are extracted from the biomass using an organic solvent.

8. A method according to claim 1, whereby the organism from which the lipids are extracted, belongs to a genus selected from the group consisting of Rhodophyta, Cyanidiophyceae, Chlorophyta, Cyanophyta, Diatoms, Phaeophyceae, Dinoflagelate, Dinophyta and Galdieria.

9. A method according to claim 1, whereby the organism from which the hydrolysate comprising amino acids is produced, is an organism selected from the group consisting of algae, fungi, yeasts and methylotrophic bacteria.

10. A, method according to claim 9, whereby the organism belongs to a genus selected from the group consisting of Pichia, Saccharoinyces, Hansenula, Cvanidiunr, Ga/dienia, Porphynidiunr, Spirzdirra, and Merhvlobacilhis.

11. A method according to claim 1, whereby the further components necessary for growth of the mammalian or insect cells comprise one or more of:

(a) one or more of glucose, fructose, and sucrose;

(b) one or more Krebs-cycles intermediates selected from the group consisting of citrate, succinate, fumarate, maleic acid, oxalate and malate;

(c) pyruvate; and

,

(d) one or more vitamins selected from the group consisting of thiamin, riboflavin, niacin, vitamin B6, folic acid, vitamin B 12, biotin, pantothenic acid, choline, paraaminobenzoic acid and alpha-tocopherol.

12. A method according to claim 1, whereby substantially all atoms in substrates that are used by the mammalian or insect cells for synthesis of biomolecules in the nutrient medium are isotopically labelled with an isotope selected from ¹⁵N;

¹³C;

²H;

¹⁵N and ¹³C;

¹⁵N and ²H;

¹³C and ²H;

or ¹⁵N, ¹³C and ²H.

13. A method for producing a biomolecule, whereby substantially all atoms in the biomolecule are isotopically labelled, the method comprising the steps of:

(a) growing a culture of mammalian or insect cells capable of producing the biomolecule under conditions conducive to the production of the biomolecule, in a nutrient medium produced in a method according to claim 1;

and

(b) recovery of the biomolecule.

14. A method according to claim 13, wherein the biomolecule is a soluble protein or a membrane protein.

15. A method according to claim 14, wherein the mammalian or insect cells capable of producing the protein comprise an expression vector comprising a nucleotide sequence coding for the protein.

16. A method according to claim 14, wherein the protein is a mammalian protein.

17. A method for obtaining structural information on a biomolecule, the method comprising the steps of:

(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.

18. A method according to claim 17, wherein the spectroscopic analysis comprises NMR spectroscopy.

19. A method according to claim 17, wherein the structural information on a biomolecule is information about the three-dimensional structure of the biomolecule.

20. A method according to claim 17, wherein the biomolecule is a protein complexed to a second biomolecule.

21. A method according to claim 20, whereby 20-100% of the hydrogen atoms in the second biomolecule are uniformly substituted with the isotope ²H.

22. A method according to claim 21, wherein the second biomolecule is a protein.

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:

(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.

24. A nutrient medium according to claim 23, whereby the source of amino acids comprises an hydrolysate comprising amino acids that is produced from yeast biomass, whereby the hydrolysis of the biomass comprises autohydrolysis.

25. A nutrient medium according to claim 23, whereby the source of lipids comprises fatty acids, steroids, and lipid soluble vitamins.

26. A nutrient medium according to claim 23, whereby the carbohydrate energy source is one or more of glucose, fructose, and sucrose;

the organic acids are one or more of pyruvate and the Krebs-cycles intermediates selected from the group consisting of citrate, succinate, fumarate, maleic acid, oxalate and malate;

the vitamins are one or more vitamins selected from the group consisting of thiamin, riboflavin, niacin, vitamin B6, folic acid, vitamin B12, biotin, pantothenic acid, choline, para-aminobenzoic acid and alpha-tocopherol.

27. A nutrient medium according to claim 23, whereby substantially all atoms in (a), (b), and (c), and, optionally in (d), (e), (f), (g) and (h) are isotopically labelled with an isotope selected from ¹⁵N;

¹³C;

²H;

¹⁵N and ¹³C;

¹⁵N and ²H;

¹³C and ²H;

or ¹⁵N, ¹³C and ²H.

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.

30. A mammalian membrane protein according to claim 28, whereby the protein is a human protein.

29. A mammalian membrane protein whereby 20-100% of the hydrogen atoms in the protein are uniformly substituted with the isotope ²H.