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US 6,472,171 B1Granted
Expression of procollagen in yeast
AssigneesCohesion Technologies, Inc., Genotypes, Inc.
InventorsDavid P. Toman, Gregory A. Daniels, Richard A. Berg, Ronald A. Hitzeman, George E. Chisholm
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Abstract
The invention discloses methods for effecting the production of recombinant mammalian procollagen in yeast, as well as compositions comprising yeast cells cap producing mammalian procollagen.
Claims25
A method for making a mammalian collagen or procollagen in yeast, the method comprising the steps of:
- (a) incubating a yeast cell comprising:
- (i) a stable genetic construct comprising in opposite orientations, first and second mammalian collagen genes operably linked to a first single divergent heterologous promoter;
- (ii) a prolyl hydroxylase gene;
- (iii) a protein disulfide isomerase gene, wherein the prolyl hydroxylase and protein disulfide isomerase genes are expressed to produce polypeptides that are associated into active proteins during the incubating step, in a medium under conditions wherein the collagen genes are expressed as procollagen chains and the chains secreted in the form of triple helical procollagen;
- and
- (b) recovering the triple helical procollagen.
3 more elements
A method for making a mammalian collagen or procollagen in yeast, the method comprising the steps of:
- (a) incubating a yeast cell comprising:
- (i) a stable genetic construct comprising in opposite orientations, first and second mammalian collagen genes operably linked to a first single divergent heterologous promoter;
- (ii) a prolyl hydroxylase gene;
- (iii) a protein disulfide isomerase gene, wherein the prolyl hydroxylase and protein disulfide isomerase genes are expressed to produce polypeptides that are associated into active proteins during the incubating step, in a medium under conditions wherein the collagen genes are expressed as procollagen chains and the chains secreted in the form of triple helical procollagen,
- wherein the yeast cell is Saccharomyces cerevisiae, the prolyl hydroxylase and protein disulfide isomerase genes are integrated into the genome of the yeast, the first single divergent heterologous promoter is a GAL1-10 promoter, the collagen genes reside on a plasmid in the yeast, the prolyl hydroxylase gene and protein disulfide isomerase genes are operably linked to a second single divergent heterologous promoter, the first and second single divergent heterologous promoters are the same, and the mammalian collagen genes are human collagen genes;
- and
- (b) recovering the triple helical procollagen.
4 more elements
A yeast cell comprising:
- (i) a stable genetic construct comprising in opposite orientations, first and second mammalian collagen genes operably linked to a first single divergent heterologous promoter;
- (ii) a prolyl hydroxylase gene;
- and (iii) a protein disulfide isomerase gene.
Dependencies are inferred from the claim text.