Compositions and methods for protein structural determinations
AssigneeMartek Biosciences Corporation
InventorJonathan M. Brown
Life of this patent
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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 .sup.2 H and .sup.13 C or triple-labeled with .sup.2 H, .sup.13 C and .sup.15 N is disclosed. The invention is also directed to a method for producing the nutrient medium.
Claims28
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 .sup.13 C or with both .sup.13 C and .sup.15 N and in which from about 20% to about 100% of the hydrogen atoms in the amino acids in such nutrient medium are .sup.2 H.
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 .sup.13 C or in which substantially all of the nitrogen in any substrate used by the microorganism for protein biosynthesis is .sup.15 N, or in which substantially all of the hydrogen in any substrate used by the microorganism for protein biosynthesis is .sup.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.
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 .sup.13 C and in which from about 20% to about 100% of the hydrogen atoms in the amino acids in such nutrient medium are .sup.2 H.
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 .sup.13 C and .sup.15 N and in which from about 20% to about 100% of the hydrogen atoms in the amino acids in such nutrient medium are .sup.2 H.
Dependencies are inferred from the claim text.