Appeal No. 94-1775
Application 07/488,513
Claims 1, 18, 15 and 16 are illustrative of the subject matter on appeal and read as
follows:
1. Method for the production of proteins with hirudin activity comprising culturing
under appropriate nutrient conditions yeast cells transformed with a hybrid vector
comprising
one to four DNA inserts each comprising a yeast promoter selected from the group
consisting of the PHO5 promoter, the GAPDH promoter 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 desulphato-hirudin which DNA segment is under transcriptional control
of said yeast promoter, and a DNA sequence containing eukaryotic transcription
termination signals,
and a selective gene marker selected from the group consisting of URA1, URA3,
ARG4, LEU2, HIS3, HIS4, TRP5 and TRP1, wherein the proteins with hirudin activity are
predominantly secreted into the culture medium, and
isolating the proteins with hirudin activity from the culture medium.
18. A method according to claim 1 wherein about 90% or more of the proteins with
hirudin activity are secreted into the culture medium.
15. A yeast hybrid vector comprising one to four DNA inserts each comprising a
yeast promoter selected from the group consisting of the PHO5 promoter, the GAPDH
promoter 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 which DNA segment is under transcriptional control of said yeast
promoter, and a DNA sequence containing eukaryotic transcription termination signals,
and a selective gene marker selected from the group consisting of URA1, URA3, ARG4,
LEU2, HIS3, HIS4, TRP5 and TRP1.
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