EP0259953A1
Expired / Lapsed
EP
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Cloning and expression of acidic fibroblast growth factor
Assignee: Merck and Co Inc
•
Filing: 1987-07-09
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Granted: —
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Expires: 2007-07-09
Abstract
Abstract Unique genes coding for the amino acid sequence of bovine acidic fibroblast growth factor (aFGF) and human aFGF are constructed. The bovine gene is derived from reverse translation of the aFGF amino acid sequence while the human gene is derived by specific point mutations of the bovine gene. Each gene construct is inserted into an expression vector which is used to transform an appropriate host. The transformed host cells produce recombinant aFGF (r-aFGF), human or bovine, which is purified and has an activity equivalent to the native protein.
Inventors
- 👤 David L. Linemeyer
- 👤 Linda J. Kelly
- 👤 Kenneth A. Thomas, Jr.
- 👤 Guillermo Gimenez-Gallego
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 16:09
CPC Classifications
C; C12; C12N; C12N15/00; C07; C07K; C07K14/00; C07K14/435; C07K14/475; C07K14/50; C07K14/501; A; A61; A61P; A61P17/00; A61P43/00; A61K; A61K38/00
Claims Analysis
Total Claims: 76
Claim 1
Independent Claim
6 words
Recombinant bovine acidic fibroblast growth factor.
Claim 2
Dependent Claim
6 words
Recombinant bovine acidic fibroblast growth factor.
Claim 3
Dependent Claim
12 words
Recombinant bovine acidic fibroblast growth factor having an amino acid sequence of:
Claim 4
Dependent Claim
12 words
Recombinant bovine acidic fibroblast growth factor having an amino acid sequence of:
Claim 5
Dependent Claim
23 words
The recombinant bovine acidic fibroblast growth factor of Claim 2 wherein there is attached to the phenylalanine at the first position a methionine.
Claim 6
Dependent Claim
9 words
Microheterogeneous forms of recombinant bovine acidic fibroblast growth factor.
Claim 7
Dependent Claim
9 words
Microheterogeneous forms of recombinant bovine acidic fibroblast growth factor.
Claim 8
Dependent Claim
10 words
A nucleotide sequence coding for bovine acidic fibroblast growth factor.
Claim 9
Dependent Claim
10 words
A nucleotide sequence coding for bovine acidic fibroblast growth factor.
Claim 10
Dependent Claim
15 words
A nucleotide sequence coding for the recombinant bovine acidic fibroblast growth factor of Claim 2.
Claim 11
Dependent Claim
15 words
A nucleotide sequence coding for the recombinant bovine acidic fibroblast growth factor of Claim 3.
Claim 12
Dependent Claim
15 words
The nucleotide sequence of Claim 6 wherein the base sequence is any of the following:
Claim 13
Dependent Claim
19 words
The nucleotide sequence of Claim 8 wherein the code for phenylalanine is preceeded by a code for the methionine.
Claim 14
Dependent Claim
11 words
The nucleotide sequence of Claim 9 wherein the base sequence is:
Claim 15
Dependent Claim
12 words
An expression plasmid comprising the nucleotide sequence of Claim 10 inserted therein.
Claim 16
Dependent Claim
13 words
The plasmid of Claim 11 wherein the structure is shown in Figure I.
Claim 17
Dependent Claim
10 words
The plasmid of Claim 11 wherein the plasmid is pBR322.
Claim 18
Dependent Claim
13 words
A host that is compatible with and contains the plasmid of Claim 11.
Claim 19
Dependent Claim
11 words
The host of Claim 14 wherein said host is E. coli.
Claim 20
Dependent Claim
16 words
The host of Claim 15 wherein said host is E. coli JM105 or E. coli DH5.
Claim 21
Dependent Claim
22 words
The plasmid of Claim 11 wherein said plasmid is capable of expressing the amino acid sequence of bovine acidic fibroblast growth factor.
Claim 22
Dependent Claim
17 words
A protein produced by the host of Claim 14 capable of stimulating DNA synthesis in responsive cells.
Claim 23
Dependent Claim
77 words
A process for the production of bovine acidic fibroblast growth factor comprising the following steps: a. providing a plasmid comprising a nucleotide sequence coding for bovine acidic fibroblast growth factor, wherein the nucleotide sequence is capable of being expressed by a host containing the plasmid; followed by b. incorporating the plasmid into the host; and c. maintaining the host containing the plasmid under conditions suitable for expression of the nucleotide sequence producing bovine acidic fibroblast growth factor.
Claim 24
Dependent Claim
77 words
A process for the production of bovine acidic fibroblast growth factor comprising the following steps: a. providing a plasmid comprising a nucleotide sequence coding for bovine acidic fibroblast growth factor, wherein the nucleotide sequence is capable of being expressed by a host containing the plasmid; followed by b. incorporating the plasmid into the host; and c. maintaining the host containing the plasmid under conditions suitable for expression of the nucleotide sequence producing bovine acidic fibroblast growth factor.
Claim 25
Dependent Claim
17 words
A process according to Claim 19, Step a, wherein the nucleotide sequence is that of Claim 10.
Claim 26
Dependent Claim
14 words
A process according to Claim 19, Step b, wherein the host is E. coli..
Claim 27
Dependent Claim
26 words
A wound healing pharmaceutical composition comprising a pharmaceutical carrier and an effective wound healing amount of the recombinant bovine acidic fibroblast growth factor of Claim 1.
Claim 28
Dependent Claim
26 words
A wound healing pharmaceutical composition comprising a pharmaceutical carrier and an effective wound healing amount of the recombinant bovine acidic fibroblast growth factor of Claim 2.
Claim 29
Dependent Claim
26 words
A wound healing pharmaceutical composition comprising a pharmaceutical carrier and an effective wound healing amount of the recombinant bovine acidic fibroblast growth factor of Claim 3.
Claim 30
Dependent Claim
35 words
A method of promoting wound healing which comprises the administration to a patient in need of such treatment of an effective wound healing amount of the recombinant bovine acidic fibroblast growth factor of Claim 2.
Claim 31
Dependent Claim
35 words
A method of promoting wound healing which comprises the administration to a patient in need of such treatment of an effective wound healing amount of the recombinant bovine acidic fibroblast growth factor of Claim 3.
Claim 32
Dependent Claim
6 words
Recombinant human acidic fibroblast growth factor.
Claim 33
Dependent Claim
6 words
Recombinant human acidic fibroblast growth factor.
Claim 34
Dependent Claim
12 words
Recombinant human acidic fibroblast growth factor having an amino acid sequence of:
Claim 35
Dependent Claim
12 words
Recombinant human acidic fibroblast growth factor having an amino acid sequence of:
Claim 36
Dependent Claim
23 words
The recombinant human acidic fibroblast growth factor of Claim 28 wherein there is attached to the phenylalanine at the first position a methionine.
Claim 37
Dependent Claim
33 words
The recombinant human acidic fibroblast growth factor of Claim 28 wherein the phenylalanine at the first position is removed and the amino acid at the second position is either asparagine or asparatic acid.
Claim 38
Dependent Claim
9 words
Microheterogeneous forms of recombinant human acidic fibroblast growth factor.
Claim 39
Dependent Claim
9 words
Microheterogeneous forms of recombinant human acidic fibroblast growth factor.
Claim 40
Dependent Claim
11 words
A nucleotide sequence coding for human recombinant acidic fibroblast growth factor.
Claim 41
Dependent Claim
11 words
A nucleotide sequence coding for human recombinant acidic fibroblast growth factor.
Claim 42
Dependent Claim
15 words
A nucleotide sequence coding for the human recombinant acidic fibroblast growth factor of Claim 29.
Claim 43
Dependent Claim
11 words
The nucleotide sequence of Claim 33 wherein the base sequence is:
Claim 44
Dependent Claim
23 words
The nucleotide sequence of Claim 10 wherein the base sequence is substituted by point mutations to give the base sequence of Claim 34.
Claim 45
Dependent Claim
12 words
An expression plasmid comprising the nucleotide sequence of Claim 34 inserted therein.
Claim 46
Dependent Claim
13 words
The plasmid of Claim 36 wherein the structure is shown in Figure I.
Claim 47
Dependent Claim
10 words
The plasmid of Claim 37 wherein the plasmid is pBR322.
Claim 48
Dependent Claim
13 words
A host that is compatible with and contains the plasmid of Claim 36.
Claim 49
Dependent Claim
9 words
A host of Claim 39 which is E. coli..
Claim 50
Dependent Claim
16 words
The host of Claim 40 wherein said host is E. coli JM105 or E. coli DH5.
Claim 51
Dependent Claim
18 words
The plasmid of Claim 36 which is capable of expressing the gene for human acidic fibroblast growth factor.
Claim 52
Dependent Claim
20 words
The plasmid of Claim 36 which is capable of expressing the synthetic nucleotide sequence for human acidic fibroblast growth factor.
Claim 53
Dependent Claim
17 words
A protein produced by the host of Claim 39 capable of stimulating DNA synthesis in responsive cells.
Claim 54
Dependent Claim
69 words
A process for the production of human aFGF comprising the following steps: a. providing a plasmid comprising a nucleotide sequence coding for . human aFGF, wherein the nucleotide sequence is capable of being expressed by a host containing the plasmid; followed by b. incorporating the plasmid into the host; and c. maintaining the host containing the plasmid under conditions suitable for expression of the nucleotide sequence producing human aFGF.
Claim 55
Dependent Claim
69 words
A process for the production of human aFGF comprising the following steps: a. providing a plasmid comprising a nucleotide sequence coding for . human aFGF, wherein the nucleotide sequence is capable of being expressed by a host containing the plasmid; followed by b. incorporating the plasmid into the host; and c. maintaining the host containing the plasmid under conditions suitable for expression of the nucleotide sequence producing human aFGF.
Claim 56
Dependent Claim
17 words
A process according to Claim 45, Step a, wherein the nucleotide sequence is that of Claim 34.
Claim 57
Dependent Claim
14 words
A process according to Claim 45, step b, wherein the host is E. coli.
Claim 58
Dependent Claim
26 words
A wound healing pharmaceutical composition comprising a pharmaceutical carrier and an effective wound healing amount of the recombinant human acidic fibroblast growth factor of Claim 27.
Claim 59
Dependent Claim
26 words
A wound healing pharmaceutical composition comprising a pharmaceutical carrier and an effective wound healing amount of the recombinant human acidic fibroblast growth factor of Claim 28.
Claim 60
Dependent Claim
26 words
A wound healing pharmaceutical composition comprising a pharmaceutical carrier and an effective wound healing amound of the recombinant human acidic fibroblast growth factor of Claim 29.
Claim 61
Dependent Claim
26 words
A wound healing pharmaceutical composition comprising a pharmaceutical carrier and an effective wound healing amount of the microheterogeneous forms of recombinant human acidic fibroblast growth factor.
Claim 62
Dependent Claim
26 words
A wound healing pharmaceutical composition comprising a pharmaceutical carrier and an effective wound healing amount of the microheterogeneous forms of recombinant human acidic fibroblast growth factor.
Claim 63
Dependent Claim
35 words
A method of promoting wound healing which comprises the administration to a patient in need of such treatment of an effective wound healing amount of the recombinant human acidic fibroblast growth factor of Claim 27.
Claim 64
Dependent Claim
36 words
A method of promoting wound healing which comprises the administration to a patient in need of such treatment of an effective wound healing amount of the recombinant human acidic fibroblast - growth factor of Claim 28.
Claim 65
Dependent Claim
35 words
A method of promoting wound healing which comprises the administration to a patient in need of such treatment of an effective wound healing amount of the recombinant human acidic fibroblast growth factor of Claim 29.
Claim 66
Independent Claim
35 words
A method of promoting wound healing which comprises the administration to a patient in need of such treatment of an effective growth promoting amount of the microheterogeneous forms of recombinant human acidic fibroblast growth factor.
Claim 67
Independent Claim
35 words
A method of promoting wound healing which comprises the administration to a patient in need of such treatment of an effective growth promoting amount of the microheterogeneous forms of recombinant human acidic fibroblast growth factor.
Claim 68
Independent Claim
58 words
A method of purifying recombinant acidic fibroblast growth factor (aFGF) in pure form comprising the following steps: a. Partial purification of recombinant aFGF by an affinity chromatography matrix and an acceptable eluant; followed by b. final purification of partially purified recombinant aFGF by reverse phase high performance liquid chromatography using an alkyl silane substrate and an acceptable eluant.
Claim 69
Independent Claim
58 words
A method of purifying recombinant acidic fibroblast growth factor (aFGF) in pure form comprising the following steps: a. Partial purification of recombinant aFGF by an affinity chromatography matrix and an acceptable eluant; followed by b. final purification of partially purified recombinant aFGF by reverse phase high performance liquid chromatography using an alkyl silane substrate and an acceptable eluant.
Claim 70
Dependent Claim
14 words
A method according to Claim 56, Step a, wherein the affinity matrix is heparin-Sepharose.
Claim 71
Dependent Claim
20 words
A method according to Claim 56, Step b, wherein the alkyl silane substrate contains between 3 and 18 carbon atoms.
Claim 72
Dependent Claim
17 words
A method according to Claim 56, Step b, wherein the alkyl silane substrate contains 4 carbon atoms.
Claim 73
Dependent Claim
15 words
A method according to Claim 56, Step a, wherein aFGF is eluted with sodium chloride.
Claim 74
Dependent Claim
27 words
6 1 . A method according to Claim 56, Step b, wherein aFGF is purified by an elution gradient consisting of an acid and an organic solvent.
Claim 75
Dependent Claim
17 words
A method according to Claim 61 wherein the acid is trifluoroacetic acid, phosphoric acid or acetic acid.
Claim 76
Dependent Claim
14 words
A method according to Claim 61 wherein the organic solvent is acetonitrile or ethanol.
References Cited (Backward Citations)
EP0131150A2
Cited
EP0225701A1
Cited
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