Patent US4766073A successfully refreshed from Google Patents.
US4766073A
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Expression of biologically active PDGF analogs in eucaryotic cells
Assignee: Zymogenetics Inc
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Filing: 1986-08-13
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Granted: 1988-08-23
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Expires: 2006-08-13
Abstract
Abstract Methods for expressing a variety of biologically active PDGF analogs in eucaryotic cells are disclosed. The methods generally comprise introducing into a eucaryotic host cell a DNA construct capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells. The DNA construct contains a transcriptional promoter followed downstream by a suitable DNA sequence. The DNA sequence may encode a protein substantially homologous to the A-chain or the B-chain of PDGF, or a portion thereof, or an A-B heterodimer. In addition, a portion of the DNA sequence may encode at least a portion of the A-chain, while another portion encodes at least a portion of the B-chain of PDGF. Eucaryotic cells transformed with these DNA constructs are also disclosed. Methods of promoting the growth of mammalian cells, comprising incubating the cells with a biologically active PDGF analog expressed by a eucaryotic host cell transformed with such a DNA construct, are also disclosed.
Inventors
- 👤 Mark J. Murray
- 👤 James D. Kelly
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 23:47
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; A; A61; A61K; A61K35/00; A61K35/12; A61K35/14; A61K35/16; C07K14/435; C07K14/475; C07K14/49; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/625; C12N15/63; C12N15/67; C12N15/74; C12N15/78; C12N15/79; C12N15/80; C12N15/81; A61K38/00; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/036; C07K2319/70; C07K2319/74; C07K2319/75; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/11011; C12N2740/11022; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/12; Y10S930/22; Y10S930/221
Claims Analysis
Total Claims: 59
Claim 1
Independent Claim
41 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.
Claim 2
Dependent Claim
41 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.
Claim 3
Dependent Claim
13 words
The vector of claim 1 wherein said eucaryotic cell is a yeast cell.
Claim 4
Dependent Claim
19 words
The vector of claim 1 wherein said protein includes at least one amino acid substitution of a cysteine residue.
Claim 5
Dependent Claim
93 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.
Claim 6
Dependent Claim
93 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.
Claim 7
Dependent Claim
13 words
The vector of claim 4 wherein said eucaryotic cell is a yeast cell.
Claim 8
Dependent Claim
28 words
The vector of claim 4 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-109.
Claim 9
Dependent Claim
36 words
The vector of claim 4 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-97 fused in reading frame A-chain amino acids 92-104.
Claim 10
Dependent Claim
19 words
The vector of claim 4 wherein said protein includes at least one amino acid substitution of a crysteine residue.
Claim 11
Dependent Claim
41 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promotor followed downstream by a DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.
Claim 12
Dependent Claim
41 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promotor followed downstream by a DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.
Claim 13
Dependent Claim
13 words
The vector of claim 9 wherein said eucaryotic cell is a yeast cell.
Claim 14
Dependent Claim
15 words
The vector of claim 9 wherein said protein is a monomeric species having biological activity.
Claim 15
Dependent Claim
19 words
The vector of claim 9 wherein said protein includes at least one amino acid substitution of a cysteine residue.
Claim 16
Dependent Claim
44 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.
Claim 17
Dependent Claim
44 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.
Claim 18
Dependent Claim
68 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein having substantially the same biological activity as PDGF, and a signal sequence positioned upstream from and in proper reading frame with said gene, said signal sequence directing the secretion of the protein from the eucaryotic cell.
Claim 19
Dependent Claim
68 words
A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein having substantially the same biological activity as PDGF, and a signal sequence positioned upstream from and in proper reading frame with said gene, said signal sequence directing the secretion of the protein from the eucaryotic cell.
Claim 20
Independent Claim
56 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.
Claim 21
Independent Claim
56 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.
Claim 22
Dependent Claim
15 words
The method of claim 15 wherein said cultured eucaryotic cell is a cultured yeast cell.
Claim 23
Dependent Claim
19 words
The method of claim 15 wherein said protein includes at least one amino acid substitution of a cysteine residue.
Claim 24
Independent Claim
108 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.
Claim 25
Independent Claim
108 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.
Claim 26
Dependent Claim
14 words
The method of claim 18 wherein said eucaryotic host cell is a yeast cell.
Claim 27
Dependent Claim
28 words
The method of claim 18 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-109.
Claim 28
Dependent Claim
36 words
The method of claim 18 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-97 fused inreading frame to A-chain amino acids 92-104.
Claim 29
Dependent Claim
19 words
The method of claim 18 wherein said protein includes at least one amino acid substitution of a cysteine residue.
Claim 30
Independent Claim
56 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.
Claim 31
Independent Claim
56 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.
Claim 32
Dependent Claim
14 words
The method of claim 23 wherein said cultured eucaryotic cell is a cultured cell.
Claim 33
Dependent Claim
15 words
The method of claim 23 wherein said protein is a monomeric species having biological activity.
Claim 34
Dependent Claim
18 words
The method of claim 23 wherein said protein includes at least one amino acid substitution of a cysteine
Claim 35
Independent Claim
59 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.
Claim 36
Independent Claim
59 words
A method of preparing biologically active PDGF analogs, comprising: introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.
Claim 37
Dependent Claim
46 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.
Claim 38
Dependent Claim
46 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.
Claim 39
Dependent Claim
17 words
The eucaryotic host cell of claim 28 wherein said cultured eucaryotic cell is a cultured yeast cell.
Claim 40
Dependent Claim
21 words
The eucaryotic host cell of claim 28 wherein said protein includes at least one amino acid substitution of a cysteine residue.
Claim 41
Dependent Claim
47 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.
Claim 42
Dependent Claim
47 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.
Claim 43
Dependent Claim
35 words
The eucaryotic host cell of claim 29 wherein said protein contains a proteolytic processing site, and said vector further encodes a prepropolypeptide lacking a proteolytic processing site, thereby resulting in proteolytic processing with the B-chain.
Claim 44
Dependent Claim
22 words
The eucaryotic host cell of claim 29 wherein said protein lacks substantially the first twenty-eight amino acids of the B-chain of PDGF.
Claim 45
Dependent Claim
17 words
The eucaryotic host cell of claim 29 wherein said protein is a monomeric species having biological activity.
Claim 46
Dependent Claim
21 words
The eucaryotic host cell of claim 29 wherein said protein includes at least one amino acid substitution of a cysteine residue.
Claim 47
Dependent Claim
99 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.
Claim 48
Dependent Claim
99 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.
Claim 49
Dependent Claim
17 words
The eucaryotic host cell of claim 34 wherein said cultured eucaryotic cell is a cultured yeast cell.
Claim 50
Dependent Claim
30 words
The eucaryotic host cell of claim 34 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-109.
Claim 51
Dependent Claim
39 words
The eucaryotic host cell of claim 34 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-97 fuse in reading frame to A-chain amino acids 92-104.
Claim 52
Dependent Claim
21 words
The eucaryotic host cell of claim 34 wherein said protein includes at least one amino acid substitution of a cysteine residue.
Claim 53
Dependent Claim
50 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.
Claim 54
Dependent Claim
50 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.
Claim 55
Dependent Claim
17 words
The eucaryotic host cell of claim 39 wherein said cultured eucaryotic cell is a cultured yeast cell.
Claim 56
Dependent Claim
80 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a prepropolypetide lacking a proteolytic processing site, said DNA sequence being followed downstream by a DNA sequence encoding a protein containing a proteolytic processing site which is substantially homologous to the B-chain of PDGF thereby resulting in proteolytic processing within the B-chain of PDGF.
Claim 57
Dependent Claim
80 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a prepropolypetide lacking a proteolytic processing site, said DNA sequence being followed downstream by a DNA sequence encoding a protein containing a proteolytic processing site which is substantially homologous to the B-chain of PDGF thereby resulting in proteolytic processing within the B-chain of PDGF.
Claim 58
Dependent Claim
58 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to a portion of the B-chain of PDGF from amino acid 29 to amino acid 109.
Claim 59
Dependent Claim
58 words
A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to a portion of the B-chain of PDGF from amino acid 29 to amino acid 109.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-08-23 | 1992-02-22 | 1992-08-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-08-23 | 1996-02-22 | 1996-08-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-08-23 | 2000-02-22 | 2000-08-23 | $7700.00 | Window Closed / Lapsed |