Compositions and methods for protein structural determinations

• US 5,627,044 A
• Filed: 09/27/1994
• Issued: 05/06/1997
• Est. Priority Date: 02/05/1993
• Status: Expired due to Fees

• Current Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Brown, Jonathan M.
• Primary Examiner(s): Kight, John
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US08/307,768
• Time in Patent Office: 952 Days
• Field of Search: 435/29, 435/113, 435/110, 435/114, 435/253.6, 435/4, 436/86, 436/15
• US Class Current: 435/29
• CPC Class Codes: C07B 59/001 Acyclic or carbocyclic compounds
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12P 13/04 Alpha- or beta- amino acids other amino acids C12P13/005
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 33/534 with radioactive label
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]

Abstract

A nutrient medium comprising amino acids and other substrates used by mammalian or insect cells in protein synthesis that are either double-labeled with ² H and ¹³ C or triple-labeled with ² H, ¹³ C and ¹⁵ N is disclosed. The invention is also directed to a method for producing the nutrient medium.

Claims

1. A nutrient media capable of supporting the growth of a mammalian or insect cell culture, which contains all amino acids that are essential for growth of the cells, an assimilable source of carbohydrate, and essential minerals and growth factors, wherein the amino acids and any other substrate used by the cells for protein synthesis are substantially labeled with ¹³ C or with both ¹³ C and ¹⁵ N and in which from about 20% to about 100% of the hydrogen atoms in the amino acids in such nutrient medium are ² H.

2. The nutrient medium of claim 1, wherein carbohydrate source is substantially labeled with ¹³ C.

3. The nutrient medium of claim 1, wherein the amino acids are substantially labeled with ¹³ C and ¹⁵ N.

4. The nutrient medium of claim 3, wherein the carbohydrate source is substantially labeled with ¹³ C.

5. The nutrient medium of claim 3, which is supplemented with cysteine which is substantially labeled with ¹³ C and ¹⁵ N and in which from about 20% to about 100% of the hydrogen atoms are ² H.

6. The nutrient medium of claim 3 which is supplemented with glutamine which is substantially labeled with ¹³ C and ¹⁵ N and in which from about 20% to about 100% of the hydrogen atoms are ² H.

7. The nutrient medium of claim 3 which is supplemented with asparagine which is substantially labeled with ¹³ C and ¹⁵ N and in which from about 20% to about 100% of the hydrogen atoms are ² H.

8. The nutrient medium of claim 1, 2, 3, 4, 5, 6, or 7, wherein the amino acids are substantially labeled with ² H.

9. The nutrient medium of claim 8, wherein the carbohydrate source is substantially labeled with ² H.

10. The nutrient medium of claim 1, wherein about 20% to about 100% of the hydrogen atoms in the carbohydrate source in such nutrient medium are ² H.

11. A method for producing a mixture of amino acids in substantially isotopically labeled form, which comprises (a) growing a culture of microorganism in a nutrient medium in which substantially all of the carbon in any substrate used by the microorganism for protein biosynthesis is ¹³ C or in which substantially all of the nitrogen in any substrate used by the microorganism for protein biosynthesis is ¹⁵ N, or in which substantially all of the hydrogen in any substrate used by the microorganism for protein biosynthesis is ² H;

(b) recovering a protein-containing fraction from the culture;

(c) hydrolyzing the protein under acidic, non-oxidizing conditions in the presence of a sulfydryl reducing agent to produce a crude mixture of amino acids;

(d) subjecting the crude mixture of amino acids to cation exchange chromatography to produce a partially purified mixture of amino acids;

(e) subjecting the partially purified mixture of amino acids to anion exchange chromatography to produce a purified mixture of amino acids; and

(f) supplementing the purified mixture of amino acids with cysteine labeled with the same isotope or isotope mixture as the nutrient medium, wherein the cysteine is added in an amount sufficient to support protein production by such mammalian or insect cells.

12. The method of claim 11, wherein the nutrient medium and the cysteine are labeled with ¹³ C.

13. The method of claim 11, wherein the nutrient medium and the cysteine are labeled with ¹⁵ N.

14. The method of claim 11, wherein the nutrient medium and the cysteine are labeled with ² H.

15. The method of claim 11, wherein the nutrient medium and the cysteine are doubly labeled with ¹³ C and ¹⁵ N.

16. The method of claim 15, wherein the nutrient medium and the cysteine are further labeled with ² H, wherein from about 20% to about 100% of the hydrogen atoms in the purified mixture of amino acids and the cysteine are ² H.

17. The method of claim 16 which further comprises supplementing the purified mixture of amino acids with glutamine which is substantially labeled with ¹³ C and ¹⁵ N and in which from about 20% to about of the hydrogen atoms are ² H.

18. The method of claim 16 which further comprises supplementing the purified mixture of amino acids with asparagine which is substantially labeled with ¹³ C and ¹⁵ N and in which from about 20% to about 100% of the hydrogen atoms are ² H.

19. The method of claim 11, wherein the microorganism is a microalgae and the nutrient medium contains as a carbon source, ¹³ C-labeled carbon dioxide.

20. The method of claim 11, wherein the microorganism is a microalgae and the nutrient medium contains a ¹⁵ N-labeled inorganic nitrogen source.

21. The method of claim 11, wherein the microorganism is a microalgae, and the nutrient medium contains, as a carbon source, ¹³ C-labeled carbon dioxide and a ¹⁵ N-labeled inorganic nitrogen source.

22. The method of claim 11, wherein the microorganism is a microalgae and substantially all of the water in the nutrient medium is ² H₂ O.

23. The method of claim 11, wherein the cation exchange resin is employed in the H⁺ form.

24. The method of claim 11, wherein the anion exchange resin is employed in the OH^- form.

25. The method of claim 23, wherein, in step (d), acidic and neutral contaminants of the protein hydrolysate are removed by eluting the cation exchange column with a acidic solution, and the amino acids are eluted from the cation exchange column with a basic solution.

26. The method of claim 25, wherein the anion exchange column is employed in the OH^- form and wherein, in step (e), basic and neutral contaminants of the protein hydrolysate are removed by eluting the anion exchange column with a basic solution, and the amino acids are eluted from the anion exchange column with an acidic solution.

27. A nutrient media capable of supporting the growth of a mammalian or insect cell culture, which contains all amino acids that are essential for growth of the cells, an assimilable source of carbohydrate, and essential minerals and growth factors, wherein the amino acids and any other substrate used by the cells for protein synthesis are substantially labeled with ¹³ C and in which from about 20% to about 100% of the hydrogen atoms in the amino acids in such nutrient medium are ² H.

28. A nutrient media capable of supporting the growth of a mammalian or insect cell culture, which contains all amino acids that are essential for growth of the cells, an assimilable source of carbohydrate, and essential minerals and growth factors, wherein the amino acids and any other substrate used by the cells for protein synthesis are substantially labeled with both ¹³ C and ¹⁵ N and in which from about 20% to about 100% of the hydrogen atoms in the amino acids in such nutrient medium are ² H.