Patent ID: 6410272
Filing Date: 2002-06-25
Classification: A61K,A61P,C07K,C12N

Abstract:
A method for the production of desulphatohirudin compounds with hirudin activity comprising the steps of:(a) culturing under appropriate nutrent conditions yeast cells transformed with a hybrid vector, the hybrid vector comprising: one to four DNA inserts each comprising a yeast promoter selected from the group consisting of a PHO5 promoter, a GAPDH promoter, or a functional fragment thereof, and a hybrid promoter comprising upstream activation sequences of the PHO5 gene and downstream promoter elements including a functional TATA box of the GAPDH gene, a DNA segment consisting of a first DNA sequence encoding the PHO5 or invertase signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin wherein the DNA segment is under transcriptional control of the yeast promoter, and a DNA sequence containing eukaryotic transcription termination signals, and a selective gene marker, wherein about 90 percent or more of the desulphatohirudin compounds are secreted into the culture medium as measured by high pressure liquid chromatography, and(b) isolating the proteins with hirudin activity from the culture medium.