EP0225633A2
Expired / Lapsed
EP
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Production of thrombin inhibitors
Assignee: UCP Gen Pharma AG
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Filing: 1986-12-09
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Granted: —
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Expires: 2006-12-09
Abstract
Abstract A method for the production and secretion of proteins with hirudin activity in an eukaryotic host organism is provided. There are also provided hybrid vectors comprising a DNA sequence encoding a signal peptide upstream of and in reading frame with the structural gene for desulphatohirudin, and eukaryotic host organisms transformed with said hybrid vectors.
Inventors
- 👤 Bernd Dr. Meyhack
- 👤 Walter Dr. Märki
- 👤 Jutta Dr. Heim
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 16:45
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2405; C12N9/2408; C12N9/2411; C12N9/2428; A; A61; A61P; A61P7/00; A61P7/02; C07; C07K; C07K14/00; C07K14/81; C07K14/815; C12N15/00; C12N15/09; C12N15/63; C12N15/70; C12N15/79; C12N15/80; C12N15/81; C12N15/87; C12N15/90; C12N9/16; A61K; A61K38/00; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/036; C07K2319/33
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
132 words
Method for the production of proteins with hirudin activity comprising culturing under appropriate nutrient conditions an eukaryotic host organism transformed with a hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, and isolating the proteins with hirudin activity from the culture medium and, if desired, isolating optionally additional protein with hirudin activity which is cell associated from the cell interior, and, if desired, converting a resulting protein with hirudin activity into a different protein with hirudin activity.
Claim 2
Dependent Claim
132 words
Method for the production of proteins with hirudin activity comprising culturing under appropriate nutrient conditions an eukaryotic host organism transformed with a hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, and isolating the proteins with hirudin activity from the culture medium and, if desired, isolating optionally additional protein with hirudin activity which is cell associated from the cell interior, and, if desired, converting a resulting protein with hirudin activity into a different protein with hirudin activity.
Claim 3
Dependent Claim
26 words
Method according to claim 1 characterized in that the expression control sequence is derived from the genomic DNA of yeast for use in a yeast host.
Claim 4
Dependent Claim
37 words
Method according to claim 1 characterized in that the expression control sequence is a hybrid promoter comprising upstream activation sequences of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene.
Claim 5
Dependent Claim
55 words
Method according to claim 1 characterized in that the DNA sequence encoding a signal peptide is selected from the group consisting of the signal sequences of the yeast invertase, a-factor, α-factor, pheromone p e ptidase, "killer toxin" and repressible acid phos- p a t ase (PHO5) genes and the glucoamylase signal sequence from Aspergillus awamori.
Claim 6
Dependent Claim
21 words
Method according to claim 1 for the production of Des-( Leu 64 , Gln 65 )-desulphatohirudin variant HVl having the formula
Claim 7
Dependent Claim
17 words
Method according to claim 1 for the production of Des-(Gln 65 )-desulphatohirudin variant HVl having the formula
Claim 8
Dependent Claim
15 words
Method according to claim 1 for the production of desulphatohirudin variant HV1 having the formula
Claim 9
Dependent Claim
15 words
Method according to claim 1 for the production of desulphatohirudin variant HV2 having the formula
Claim 10
Dependent Claim
15 words
Method according to claim 1 for the production of desulphatohirudin variant PA having the formula
Claim 11
Dependent Claim
10 words
Method according to claim 1 for the production of des-(Val)2-desulphatohirudin.
Claim 12
Dependent Claim
58 words
A DNA construct containing an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence .encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals.
Claim 13
Dependent Claim
58 words
A DNA construct containing an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence .encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals.
Claim 14
Dependent Claim
18 words
A construct according to claim 11 characterized in that the expression control sequence is derived from a virus.
Claim 15
Dependent Claim
21 words
A construct according to claim 11 characterized in that the expression control sequence is derived from the genomic DNA of yeast.
Claim 16
Dependent Claim
50 words
A construct according to claim 11 characterized in that the DNA sequence encoding a signal peptide is selected from the group consisting of the signal sequences of the yeast invertase, a-factor, a-factor, pheromone peptidase, "killer toxin" and repressible acid phosphatase (PH05) genes and the glucoamylase signal sequence from Aspergillus awamori.
Claim 17
Dependent Claim
53 words
A construct according to claim 11 containing the PHO5 promoter, a DNA segment consisting of the PH05 signal sequence upstream of and in reading frame with a DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of the PH05 promoter, and the 3' flanking sequence of the PHO5 gene.
Claim 18
Dependent Claim
53 words
A construct according to claim 11 containing the GAPDH promoter, a DNA segment consisting of the PH05 signal sequence upstream of and in reading frame with a DNA sequence coding for mature desulphatohirudin, which DNA segment is under transcriptional control of the GAPDH promoter, and the 3' flanking sequence of the PH05 gene.
Claim 19
Dependent Claim
77 words
A construct according to claim 11 containing a hybrid promoter comprising upstream activation site(s) of the yeast PH0 5 gene and a downstream promoter element including the TATA box of the yeast GAPDH gene, a DNA segment consisting of the PH05 signal sequence upstream of and in reading frame with a DNA sequence coding for mature desulphatohirudin, which DNA segment is under transcriptional control of the hybrid promoter, and the 3' flanking sequence of the PH05 gene.
Claim 20
Dependent Claim
55 words
A construct according to claim 11 containing the PH 05 promoter, a DNA segment consisting of the invertase signal sequence upstream of and in reading frame with a DNA sequence coding for mature desulphatohirudin which D NA segment is under transcriptional control of the PH05 promoter, and the 3' flanking sequence of the PH05 gene.
Claim 21
Dependent Claim
79 words
Method for the preparation of a DNA construct according to claim 11 comprising linking an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for desulphatohirudin, and a DNA sequence containing eukaryotic transcription termination signals in such a way that proper expression of the DNA segment and secretion of the produced desulphatohirudin is effected in an eukaryotic host organism.
Claim 22
Dependent Claim
67 words
A hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a D N A segment consisting of a first DNA sequence encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals.
Claim 23
Dependent Claim
67 words
A hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a D N A segment consisting of a first DNA sequence encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals.
Claim 24
Dependent Claim
20 words
A hybrid vector according to claim 20 containing a DNA construct according to any one of claim 11 to 18.
Claim 25
Dependent Claim
96 words
Method for the preparation of a hybrid vector according to claim 20 comprising introducing one or multiple DNA constructs each containing an eukaryotic epression control sequence, a DNA segment consisting of a first DNA sequence encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, as such or introducing the components of said DNA constructs successively in the predetermined order into a vector DN A .
Claim 26
Dependent Claim
75 words
A eukaryotic host organism transformed with a hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first D NA sequence encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, and mutants thereof.
Claim 27
Dependent Claim
75 words
A eukaryotic host organism transformed with a hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first D NA sequence encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, and mutants thereof.
Claim 28
Dependent Claim
22 words
A eukaryotic host organism according to claim 23 transformed with a hybrid vector according to any one of claims 20 and 21.
Claim 29
Dependent Claim
17 words
A transformed eukaryotic host organism according to claim 23 selected from a mammalian cell line and yeast.
Claim 30
Dependent Claim
95 words
Method for the production of a transformed eukaryotic host organism according to claim 23 comprising transforming eukaryotic host cells with a hybrid vector having one or multiple DNA inserts each comprising a eukaryotic expression control sequence, a DNA segment consisting of a st DNA sequence encoding a 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 expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, and, if desired, mutating the transformed eukaryotic host cells obtained.
Claim 31
Dependent Claim
5 words
Des-(Gln 65 )-desulphatohirudin variant HVl.
Claim 32
Dependent Claim
5 words
Des-(Gln 65 )-desulphatohirudin variant HVl.
References Cited (Backward Citations)
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