US4769328A
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Expression of biologically active PDGF analogs in yeast
Assignee: Zymogenetics Inc
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Filing: 1984-10-12
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Granted: 1988-09-06
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Expires: 2004-10-12
Abstract
Abstract Biologically active PDGF analogs expressed in yeast are disclosed. The analogs are produced by yeast strains transformed with an extrachromosomal element composed of a strong transcriptional promoter directing the expression of a gene which encodes a protein having substantially the same biological activity as PDGF. Suitable genes include the v-sis gene or a derivative of the v-sis gene of simian sarcoma virus or portions thereof, or the human cDNA gene for PDGF or portions thereof. A secretory signal sequence may be provided upstream of the gene, enabling secretion of the gene product from the yeast host cell. Mitogenic activity is one of the biological activities possessed by these PDGF analogs, making them useful in promoting the growth of mammalian cells.
Inventors
- 👤 Mark J. Murray
- 👤 James D. Kelly
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 08:25
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/78; A; A61; A61K; A61K35/00; A61K35/12; A61K35/14; A61K35/16; C12N15/67; A61K38/00; C07; C07K; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/036; C07K2319/70; C07K2319/74; C07K2319/75; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/12
Claims Analysis
Total Claims: 60
Claim 1
Independent Claim
42 words
A vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.
Claim 2
Dependent Claim
42 words
A vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.
Claim 3
Dependent Claim
20 words
The vector of claim 1 wherein said promoter is the yeast alpha-factor promoter or the yeast triose phosphate isomerase promoter.
Claim 4
Dependent Claim
21 words
The vector of claim 1 wherein said promoter is followed downstream by the signal sequence of the yeast mating pheromone alpha-factor.
Claim 5
Dependent Claim
19 words
The vector of claim 1 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof.
Claim 6
Dependent Claim
22 words
The vector of claim 1 wherein said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof.
Claim 7
Dependent Claim
18 words
The vector of claim 1 wherein said gene is the human cDNA gene for PDGF or portions thereof.
Claim 8
Dependent Claim
17 words
The vector of claim 1 wherein said gene is followed downstream by a triose phosphate isomerase terminator.
Claim 9
Dependent Claim
17 words
The vector of claim 6 wherein said human gene is derived from mRNA isolated from endothelial cells.
Claim 10
Dependent Claim
13 words
The vector of claim 1 wherein said protein is a disulfide-bonded dimeric protein.
Claim 11
Dependent Claim
64 words
A vector capable of replication in yeast and containing the yeast triose phosphate isomerase promoter, said yeast promoter being followed downstream by the signal sequence of the yeast mating pheromone alpha-factor, said signal sequence being followed downstream respectively by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, and a triose phosphate isomerase terminator.
Claim 12
Dependent Claim
64 words
A vector capable of replication in yeast and containing the yeast triose phosphate isomerase promoter, said yeast promoter being followed downstream by the signal sequence of the yeast mating pheromone alpha-factor, said signal sequence being followed downstream respectively by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, and a triose phosphate isomerase terminator.
Claim 13
Independent Claim
76 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a yeast host a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF; growing said yeast host in an appropriate medium; and isolating the protein product of said gene from said yeast host.
Claim 14
Independent Claim
76 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a yeast host a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF; growing said yeast host in an appropriate medium; and isolating the protein product of said gene from said yeast host.
Claim 15
Dependent Claim
20 words
The method of claim 11 wherein said promoter is the yeast alpha-factor promoter or the yeast triose phosphate isomerase promoter.
Claim 16
Dependent Claim
21 words
The method of claim 12 wherein said promoter is followed downstream by the signal sequence of the yeast mating pheromone alpha-factor.
Claim 17
Dependent Claim
19 words
The method of claim 13 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof.
Claim 18
Dependent Claim
22 words
The method of claim 14 wherein said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof.
Claim 19
Dependent Claim
18 words
The method of claim 15 wherein said gene is the human cDNA gene for PDGF or portions thereof.
Claim 20
Dependent Claim
17 words
The method of claim 16 wherein said human gene is derived from mRNA isolated from endothelial cells.
Claim 21
Dependent Claim
17 words
The method of claim 17 wherein said gene is followed downstream by a triose phosphate isomerase terminator.
Claim 22
Dependent Claim
24 words
The method of claim 11, including after isolation of said protein product, purifying said product by gel filtration, ion exchange chromatography of affinity chromatography.
Claim 23
Dependent Claim
23 words
The method of claim 19 wherein said medium need not contain antibiotics or heavy metals, and need not be depleted of specific nutrients.
Claim 24
Dependent Claim
13 words
The method of claim 19 wherein said protein is a disulfide-bonded dimeric protein.
Claim 25
Dependent Claim
47 words
A yeast cell transformed with a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.
Claim 26
Dependent Claim
47 words
A yeast cell transformed with a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.
Claim 27
Dependent Claim
21 words
The yeast cell of claim 22 wherein said promoter is the yeast alpha-factor promoter or the yeast triose phosphate isomerase promoter.
Claim 28
Dependent Claim
22 words
The yeast cell of claim 23 wherein said promoter is followed downstream by the signal sequence of the yeast mating pheromone alpha-factor.
Claim 29
Dependent Claim
20 words
The yeast cell of claim 24 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof.
Claim 30
Dependent Claim
23 words
The yeast cell of claim 25 wherein said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof.
Claim 31
Dependent Claim
19 words
The yeast cell of claim 26 wherein said gene is the human cDNA gene for PDGF or portions thereof.
Claim 32
Dependent Claim
18 words
The yeast cell of claim 27 wherein said human gene is derived from mRNA isolated from endothelial cells.
Claim 33
Dependent Claim
14 words
The yeast cell of claim 28 wherein said protein is a disulfide-bonded dimeric protein.
Claim 34
Dependent Claim
13 words
The yeast cell of claim 29 wherein said vector is the plasmid p117-2.
Claim 35
Dependent Claim
13 words
The yeast cell of claim 30 wherein said vector is the plasmid YEpVSα.
Claim 36
Dependent Claim
13 words
The yeast cell of claim 31 wherein said vector is the plasmid YEpVS2α.
Claim 37
Dependent Claim
18 words
The yeast cell of claim 32 wherein said gene is followed downstream by a triose phosphate isomerase terminator.
Claim 38
Independent Claim
68 words
A method of promoting the growth of mammalian cells, comprising incubating the cells with a biologically active PDGF analog expressed by a yeast cell transformed with a vector capable of replication in yeast said vector containing a yeast promoter capable of directing the expression of the PDGF analog, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.
Claim 39
Independent Claim
68 words
A method of promoting the growth of mammalian cells, comprising incubating the cells with a biologically active PDGF analog expressed by a yeast cell transformed with a vector capable of replication in yeast said vector containing a yeast promoter capable of directing the expression of the PDGF analog, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.
Claim 40
Dependent Claim
13 words
The method of claim 34 wherein said growth is carried out in vitro.
Claim 41
Dependent Claim
14 words
The method of claim 34 wherein said cells are normal cells or transformed cells.
Claim 42
Dependent Claim
13 words
The method of claim 34 wherein said protein is a disulfide-bonded dimeric protein.
Claim 43
Dependent Claim
3 words
The plasmid p117-2.
Claim 44
Dependent Claim
3 words
The plasmid p117-2.
Claim 45
Dependent Claim
3 words
The plasmid YEpVSα.
Claim 46
Dependent Claim
3 words
The plasmid YEpVSα.
Claim 47
Dependent Claim
3 words
The plasmid YEpVS2α.
Claim 48
Dependent Claim
3 words
The plasmid YEpVS2α.
Claim 49
Dependent Claim
66 words
A vector capable of replication in yeast and containing the yeast triose phosphate isomerase promoter, said yeast promoter being followed downstream by the signal sequence of the yeast mating pheromone alpha-factor, said signal sequence being followed downstream respectively by the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having substantially the same biological activity as PDGF, and a triose phosphate isomerase terminator.
Claim 50
Dependent Claim
66 words
A vector capable of replication in yeast and containing the yeast triose phosphate isomerase promoter, said yeast promoter being followed downstream by the signal sequence of the yeast mating pheromone alpha-factor, said signal sequence being followed downstream respectively by the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having substantially the same biological activity as PDGF, and a triose phosphate isomerase terminator.
Claim 51
Dependent Claim
13 words
The vector of claim 13 wherein said protein is a disulfide-bonded dimeric protein.
Claim 52
Dependent Claim
61 words
A vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF.
Claim 53
Dependent Claim
61 words
A vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF.
Claim 54
Dependent Claim
13 words
The vector of claim 15 wherein said protein is a disulfide-bonded dimeric protein.
Claim 55
Independent Claim
95 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a yeast host a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF; growing said yeast host in an appropriate medium; and isolating the protein product of said gene from said yeast host.
Claim 56
Independent Claim
95 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a yeast host a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF; growing said yeast host in an appropriate medium; and isolating the protein product of said gene from said yeast host.
Claim 57
Dependent Claim
13 words
The method of claim 32 wherein said protein is a disulfide-bonded dimeric protein.
Claim 58
Dependent Claim
66 words
A yeast cell transformed with a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF.
Claim 59
Dependent Claim
66 words
A yeast cell transformed with a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF.
Claim 60
Dependent Claim
14 words
The yeast cell of claim 10 wherein said protein is a disulfide-bonded dimeric protein.
References Cited (Backward Citations)
US4350687A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-09-06 | 1992-03-07 | 1992-09-06 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-09-06 | 1996-03-07 | 1996-09-06 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-09-06 | 2000-03-07 | 2000-09-06 | $7700.00 | Window Closed / Lapsed |