US4742004A
Expired / Lapsed
US
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Method for producing enzymatically active eucaryotic sod in bacteria
Assignee: Savient Pharmaceuticals Inc
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Filing: 1984-08-27
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Granted: 1988-05-03
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Expires: 2004-08-27
Abstract
Abstract A method of producing enzymatically active eucaryotic superoxide dismutase or an analog thereof in a bacterial cell which contains and is capable of expressing a DNA sequence encoding the superoxide dismutase or analog thereof comprising maintaining the bacterial cell under suitable conditions and in a suitable production medium. The production medium is supplemented with an amount of Cu++ so that the concentration of Cu++ in the medium is greater than about about 2 ppm. The invention also concerns methods of recovering purified enzymatically active eucaryotic superoxide dismutase or analogs from bacterial cells producing the superoxide dismutase or analog.
Inventors
- 👤 Jacob R. Hartman
- 👤 Dov Kanner
- 👤 Daniel Bartfeld
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 10:00
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0089; A; A23; A23K; A23K20/00; A23K20/10; A23K20/168; A61; A61K; A61K38/00; A61K38/16; A61K38/43; A61K38/44; A61K38/446; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/61; C07K14/775; C12N15/00; C12N15/09; C12N15/63; C12N15/67; C12N15/69; C12N15/70; C12N15/73; C12Y; C12Y115/00; C12Y115/01; C12Y115/01001
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
124 words
A method of producing an enzymatically active polypeptide analog of human Cu/Zn superoxide dismutase having the identical amino acid sequence and activity of naturally occurring human Cu/Zn superoxide dismutase which comprises: growing a culture of bacterial cells in a zinc containing production medium supplemented with a nongrowth inhibiting amount of Cu ++ such that the final concentration of Cu ++ in the medium is greater than 2 ppm, wherein said cells contain and are capable of expressing DNA encoding the polypeptide analog of human Cu/Zn superoxide dismutase and wherein said culture is grown under suitable conditions such that the DNA is expressed and the polypeptide is produced in the bacterial cells; and recovering the enzymatically active analog of human Cu/Zn superoxide dismutase so produced.
Claim 2
Independent Claim
124 words
A method of producing an enzymatically active polypeptide analog of human Cu/Zn superoxide dismutase having the identical amino acid sequence and activity of naturally occurring human Cu/Zn superoxide dismutase which comprises: growing a culture of bacterial cells in a zinc containing production medium supplemented with a nongrowth inhibiting amount of Cu ++ such that the final concentration of Cu ++ in the medium is greater than 2 ppm, wherein said cells contain and are capable of expressing DNA encoding the polypeptide analog of human Cu/Zn superoxide dismutase and wherein said culture is grown under suitable conditions such that the DNA is expressed and the polypeptide is produced in the bacterial cells; and recovering the enzymatically active analog of human Cu/Zn superoxide dismutase so produced.
Claim 3
Dependent Claim
15 words
A method as in claim 1, wherein the bacterial cell is an Escherichia coli cell.
Claim 4
Dependent Claim
36 words
A method as in claim 1 wherein said culture of bacterial cells has been transformed with a plasmid, wherein said plasmid contains the DNA sequence encoding said polypeptide analog of human Cu/Zn superoxide dismutase incorporated therein.
Claim 5
Dependent Claim
15 words
A method as in claim 1, wherein said production medium is a casein hydrolysate medium.
Claim 6
Dependent Claim
13 words
A method as in claim 1, wherein said production medium is LB medium.
Claim 7
Dependent Claim
19 words
A method as in claim 1, wherein said Cu ++ concentration is from about 50 to about 250 ppm.
Claim 8
Dependent Claim
15 words
A method as in claim 4, wherein said Cu ++ concentration is about 200 ppm.
Claim 9
Dependent Claim
15 words
A method as in claim 5, wherein said Cu ++ concentration is about 75 ppm.
Claim 10
Dependent Claim
35 words
A method as in claim 1, wherein said production medium is also supplemented with an amount of Zn ++ so that the concentration of Zn ++ in the medium is greater than about 2 ppm.
Claim 11
Dependent Claim
30 words
A method as in claim 9, wherein said production medium is supplemented with about 0.8 g/l CuSO 4 .5H 2 O and about 10 mg/l ZnSO 4 .7H 2 O.
Claim 12
Dependent Claim
24 words
A method as in claim 3, wherein said plasmid is pSODβ 1 , pSODα2, pSODβ 1 T11, pSODβ 1 -BA2 or pSODβ 1 TT-1.
Claim 13
Dependent Claim
20 words
A method as in claim 1, wherein said bacterial cell is Escherichia coli strain A2097 containing plasmid pSODα2 ATCC 39786.
Claim 14
Dependent Claim
115 words
A method of recovering purified enzymatically active human Cu/Zn superoxide dismutase analog of claim 1 which comprises: (a) isolating the bacterial cells from the production medium; (b) disrupting the bacterial cells so as to form a suspension comprising cell debris and a protein supernatant solution; (c) separating said cell debris from the soluble protein supernatant solution; (d) heating said separated protein supernatant at about 65° C. for about 2 hours; (e) cooling the heated protein supernatant; (f) separating the resulting protein supernatant so as to produce a clear supernatant protein solution containing the superoxide dismutase thereof analog thereof; and (g) recovering the enzymatically active human Cu/Zn superoxide dismutase analog from the clear supernatant protein solution.
Claim 15
Dependent Claim
66 words
The method of claim 13, wherein the recovery of the enzymatically active human Cu/Zn superoxide dismutase analog from the clear supernatant protein solution further comprises: (a) treating the clear supernatant protein solution to obtain a concentrated solution of the superoxide dismutase analog; (b) subjecting the concentrated solution to both anion and cation exchange chromatography; and (c) collecting the resulting fractions containing purified superoxide dismutase analog thereof.
Claim 16
Independent Claim
128 words
A method of producing an enzymatically active polypeptide analog of human Cu/Zn superoxide dismutase having the identical amino acid sequence and activity of naturally occurring human Cu/Zn superoxide dismutase which comprises: growing a culture of cells of a strain of E. coli in a zinc containing production medium supplemented with an amount of Cu ++ such that the final concentration of Cu ++ in the medium is greater than 2 ppm wherein said cells contain a plasmid capable of expressing DNA encoding a polypeptide analog of human Cu/Zn superoxide dismutase, and wherein said plasmid is selected from the group consisting of pSODβ 1 T 11 , pSODβ 1 , pSODβ 1 TT-1 and pSODβ 1 -BA2; and recovering said polypeptide analog from the E. coli cells so cultured.
Claim 17
Independent Claim
128 words
A method of producing an enzymatically active polypeptide analog of human Cu/Zn superoxide dismutase having the identical amino acid sequence and activity of naturally occurring human Cu/Zn superoxide dismutase which comprises: growing a culture of cells of a strain of E. coli in a zinc containing production medium supplemented with an amount of Cu ++ such that the final concentration of Cu ++ in the medium is greater than 2 ppm wherein said cells contain a plasmid capable of expressing DNA encoding a polypeptide analog of human Cu/Zn superoxide dismutase, and wherein said plasmid is selected from the group consisting of pSODβ 1 T 11 , pSODβ 1 , pSODβ 1 TT-1 and pSODβ 1 -BA2; and recovering said polypeptide analog from the E. coli cells so cultured.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1991-05-03 | 1991-11-02 | 1992-05-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-05-03 | 1995-11-02 | 1996-05-03 | $3760.00 | Window Closed / Lapsed |
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