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US 7,842,477 B2Granted
Methods for producing gamma-carboxylated proteins
AssigneeAstraZeneca AB
InventorsChristel Fenge, Ann Lövgren, Anders Thelin
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Abstract
The present invention relates to methods and tools for producing large quantities of gamma-carboxylated protein comprising: (i) culturing a cell adapted to express a protein which requires gamma-carboxylation and γ-glutamyl carboxylase in a ratio of at least 10:1, under conditions suitable for expression of both proteins, and (ii) isolating gamma-carboxylated protein.
Claims67
A method for producing a protein, the method comprising:
- (a) providing a recombinant host cell containing a first nucleic acid comprising a first promoter operably linked to a nucleotide sequence encoding a protein requiring gamma-carboxylation and a second heterologous nucleic acid comprising a second promoter operably linked to a nucleotide sequence encoding a γ-glutamyl carboxylase, wherein the first promoter is stronger than the second promoter;
- (b) culturing the cell under conditions suitable for expressing both nucleic acids, wherein the protein requiring gamma-carboxylation and the γ-glutamyl carboxylase are expressed in a ratio of at least 10:1 and the protein requiring gamma-carboxylation is carboxylated in the cell, thereby producing a gamma-carboxylated protein;
- (c) isolating the gamma-carboxylated protein or an activated form thereof;
- and
- (d) preparing a pharmaceutical composition comprising the isolated gamma-carboxylated protein or an activated form thereof.
2 more elements
A method for producing a protein, the method comprising:
- (a) providing a genetically modified cell that expresses a first DNA encoding a protein requiring gamma-carboxylation and a second DNA encoding a γ-glutamyl carboxylase;
- (b) culturing the cell under conditions suitable for expressing both DNAs, wherein the protein requiring gamma-carboxylation is expressed and gamma-carboxylated in the cell, thereby producing a gamma-carboxylated protein;
- and
- (c) isolating the gamma-carboxylated protein or an activated form thereof,
- wherein the level of γ-glutamyl carboxylase mRNA expressed in the genetically modified cell is (i) higher than the level of γ-glutamyl carboxylase mRNA expressed under identical conditions in a second cell identical to the genetically modified cell except that it has not been genetically modified, and (ii) less than or equal to 10% of the level of mRNA encoding the protein requiring gamma-carboxylation expressed in the genetically modified cell.
2 more elements
A method for producing a gamma-carboxylated protein, the method comprising:
- (a) providing a cell containing a first DNA encoding a human protein requiring gamma-carboxylation and a second DNA encoding a γ-glutamyl carboxylase, wherein each of the first and second DNAs was introduced into the cell, or an ancestor of the cell, by recombinant technology;
- (b) culturing the cell under conditions suitable for expressing both DNAs, wherein the protein requiring gamma-carboxylation is expressed and gamma-carboxylated in the cell, thereby producing a gamma-carboxylated protein;
- and
- (c) isolating the gamma-carboxylated protein,
- wherein the level of γ-glutamyl carboxylase mRNA expressed in the cell is less than or equal to 10% of the level of mRNA expressed from the first DNA.
2 more elements
A method for producing a protein, the method comprising:
- (a) providing a recombinant host cell containing a first nucleic acid comprising a first promoter operably linked to a nucleotide sequence encoding a protein requiring gamma-carboxylation and a second heterologous nucleic acid comprising a second promoter operably linked to a nucleotide sequence encoding a γ-glutamyl carboxylase, wherein the first promoter is stronger than the second promoter;
- (b) culturing the cell under conditions suitable for expressing both nucleic acids, wherein the protein requiring gamma-carboxylation and the γ-glutamyl carboxylase are expressed in a ratio of at least 10:1 and the protein requiring gamma-carboxylation is carboxylated in the cell, thereby producing a gamma-carboxylated protein;
- and
- (c) isolating the gamma-carboxylated protein or an activated form thereof.
1 more element
Dependencies are inferred from the claim text.