US5089397A
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Superior mammalian expression system
Assignee: Scios LLC
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Filing: 1989-06-14
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Granted: 1992-02-18
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Expires: 2009-06-14
Abstract
Abstract An expression system for recombinant production of a desired protein comprises CHO cells transformed with a DNA sequence having the desired protein coding sequence under control of the human metallothionein-II promoter. The cells can be maintained on serum-free medium and induced in the presence of an induction mediator. In addition, the system may include an enhancer element and/or a resistance-conferring gene to provide increased levels of expression. The system can process genomic as well as intronless DNA, and is capable of producing proteins which have the same characteristics as those obtained from native sources. Human growth hormone which is indistinguishable from that produced by the pituitary has been thus produced.
Inventors
- 👤 Peter J. Kushner
- 👤 Claire L. Cofer
- 👤 Jeffrey S. Friedman
- 👤 Karen D. Talmadge
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 05:56
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6478; C12N9/6486; C07; C07K; C07K14/00; C07K14/435; C07K14/475; C07K14/505; C07K14/575; C07K14/58; C07K14/61; C07K14/775; C07K14/785; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12Y; C12Y304/00; C12Y304/23; C12Y304/23015
Claims Analysis
Total Claims: 41
Claim 1
Independent Claim
77 words
A regulatable expression system for a desired coding sequence, which system comprise Chinese Hamster Ovary (CHO) host cells in defined medium, which cells have been transformed with a DNA sequence comprising the metallothionein II (MT-II) promoter operably linked to said desired coding sequence, said medium being free of serum and further containing non-toxic metal ions and an induction mediator selected from the group consisting of iron ion and an iron-containing protein which is not a protein supplement.
Claim 2
Dependent Claim
77 words
A regulatable expression system for a desired coding sequence, which system comprise Chinese Hamster Ovary (CHO) host cells in defined medium, which cells have been transformed with a DNA sequence comprising the metallothionein II (MT-II) promoter operably linked to said desired coding sequence, said medium being free of serum and further containing non-toxic metal ions and an induction mediator selected from the group consisting of iron ion and an iron-containing protein which is not a protein supplement.
Claim 3
Dependent Claim
18 words
The system of claim 1 wherein the DNA sequence further includes an enhancer operably linked to the promoter.
Claim 4
Dependent Claim
12 words
The system of claim 2 wherein the enhancer is a viral enhancer.
Claim 5
Dependent Claim
12 words
The system of claim 3 wherein the enhancer is derived from SV40.
Claim 6
Dependent Claim
17 words
The system of claim 1 wherein the cells have been cotransformed with an amplifiable toxin-resistance conferring gene.
Claim 7
Dependent Claim
33 words
The system of claim 5 wherein the coding sequence encodes a protein selected from the group consisting of alveolar surfactant protein (ASP), human growth hormone (hGH), atrial natriuretic factor (ANF), and erythropoietin (Epo).
Claim 8
Dependent Claim
14 words
The system of claim 5 wherein the toxin-resistance conferring gene is the MT gene.
Claim 9
Dependent Claim
14 words
The system of claim 7 wherein the transformed cells are resistant to cadmium ion.
Claim 10
Dependent Claim
15 words
The system of claim 8 wherein the cells are resistant to 20 μm cadmium ion.
Claim 11
Dependent Claim
17 words
The system of claim 2 wherein the cells have been cotransformed with an amplifiable toxin-resistance conferring gene.
Claim 12
Dependent Claim
14 words
The system of claim 10 wherein the toxin-resistance conferring gene is the MT gene.
Claim 13
Dependent Claim
14 words
The system of claim 11 wherein the transformed cells are resistant to cadmium ion.
Claim 14
Dependent Claim
15 words
The system of claim 12 wherein the cells are resistant to 20 μm cadmium ion.
Claim 15
Dependent Claim
33 words
The system of claim 10 wherein the coding sequence encodes a protein selected from the group consisting of alveolar surfactant protein (ASP), human growth hormone (hGH), atrial natriuretic factor (ANF), and erythropoietin (Epo).
Claim 16
Dependent Claim
33 words
The system of claim 2 wherein the coding sequence encodes a protein selected from the group consisting of alveolar surfactant protein (ASP), human growth hormone (hGH), atrial natriuretic factor (ANF), and erythropoietin (Epo).
Claim 17
Dependent Claim
11 words
The system of claim 1 wherein the MT-II is human MT-II.
Claim 18
Dependent Claim
14 words
The system of claim 1 wherein the induction mediator is iron ion or transferrin.
Claim 19
Dependent Claim
24 words
The system of claim 1 wherein the non-toxic metal ions are zinc ions in a concentration range of 2×10 -5 to 2×10 -4 M.
Claim 20
Dependent Claim
33 words
The system of claim 1 wherein the coding sequence encodes a protein selected from the group consisting of alveolar surfactant protein (ASP), human growth hormone (hGH), atrial natriuretic factor (ANF), and erythropoietin (Epo).
Claim 21
Dependent Claim
12 words
The system of claim 19 wherein the transformed host cells are CBI-37.
Claim 22
Dependent Claim
70 words
A regulatable system for expression of a desired coding sequence which comprises mammalian host cells in serum-free medium, which cells have been transformed with a DNA sequence comprising the MT-II promoter operably linked to a desired coding sequence, wherein said serum-free medium is supplemented with an effective amount of an induction mediator selected from the group consisting of iron ion and an iron-containing protein which is not a protein supplement.
Claim 23
Dependent Claim
70 words
A regulatable system for expression of a desired coding sequence which comprises mammalian host cells in serum-free medium, which cells have been transformed with a DNA sequence comprising the MT-II promoter operably linked to a desired coding sequence, wherein said serum-free medium is supplemented with an effective amount of an induction mediator selected from the group consisting of iron ion and an iron-containing protein which is not a protein supplement.
Claim 24
Dependent Claim
20 words
The system of claim 21 wherein the induction mediator is >5 μg/ml human transferrin or 1-3×10 -5 M iron ion.
Claim 25
Dependent Claim
17 words
The system of claim 21 wherein the cells have been cotransformed with an amplifiable toxin-resistance conferring gene.
Claim 26
Dependent Claim
14 words
The system of claim 23 wherein the toxin-resistance conferring gene is the MT gene.
Claim 27
Dependent Claim
14 words
The system of claim 21 wherein the induction mediator is iron ion or transferrin.
Claim 28
Dependent Claim
18 words
The system of claim 21 wherein the DNA sequence further includes an enhancer operably linked to the promoter.
Claim 29
Dependent Claim
12 words
The system of claim 26 wherein the enhancer is a viral enhancer.
Claim 30
Dependent Claim
17 words
The system of claim 26 wherein the cells have been cotransformed with an amplifiable toxin-resistance conferring gene.
Claim 31
Dependent Claim
14 words
The system of claim 28 wherein the toxin-resistance conferring gene is the MT gene.
Claim 32
Independent Claim
78 words
A method for obtaining regulatable expression of a desired coding sequence in mammalian host cells, said method comprising inducing the host cells which have been transformed with a DNA sequence comprising the MT-II promoter operably linked to the desired coding sequence, in serum-free medium with an effective amount of an induction mediator selected from the group consisting of iron ion and an iron-containing protein which is not a protein supplement, by adding an effective amount of zinc ion.
Claim 33
Independent Claim
78 words
A method for obtaining regulatable expression of a desired coding sequence in mammalian host cells, said method comprising inducing the host cells which have been transformed with a DNA sequence comprising the MT-II promoter operably linked to the desired coding sequence, in serum-free medium with an effective amount of an induction mediator selected from the group consisting of iron ion and an iron-containing protein which is not a protein supplement, by adding an effective amount of zinc ion.
Claim 34
Dependent Claim
18 words
The method of claim 30 wherein the DNA sequence further includes an enhancer operably linked to the promoter.
Claim 35
Dependent Claim
18 words
The method of claim 31 wherein the host cells have been cotransformed with an amplifiable toxin resistance-conferring gene.
Claim 36
Dependent Claim
18 words
The method of claim 30 wherein the host cells have been cotransformed with an amplifiable toxin resistance-conferring gene.
Claim 37
Dependent Claim
19 words
The method of claim 30 wherein said cells are induced by addition of an effective amount of zinc ion.
Claim 38
Dependent Claim
14 words
The method of claim 30 wherein the induction mediator is iron ion or transferrin.
Claim 39
Dependent Claim
77 words
A regulatable system for expression of a desired coding sequence which comprises mammalian host cells transformed with a DNA sequence comprising the MT-II promoter operably linked to a desired coding sequence, in contact with a serum-free medium supplemented with an effective amount of an induction mediator selected from the group consisting of iron ion and an iron-containing protein which is not a protein supplement, and zinc ion in the range of 2×10 -5 to 2×10 -4 M.
Claim 40
Dependent Claim
77 words
A regulatable system for expression of a desired coding sequence which comprises mammalian host cells transformed with a DNA sequence comprising the MT-II promoter operably linked to a desired coding sequence, in contact with a serum-free medium supplemented with an effective amount of an induction mediator selected from the group consisting of iron ion and an iron-containing protein which is not a protein supplement, and zinc ion in the range of 2×10 -5 to 2×10 -4 M.
Claim 41
Dependent Claim
14 words
The system of claim 36 wherein the induction mediator is iron ion or transferrin.
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) | 1995-02-17 | 1995-08-19 | 1996-02-18 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-02-17 | 1999-08-19 | 2000-02-18 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-02-17 | 2003-08-19 | 2004-02-18 | $7700.00 | Window Closed / Lapsed |