US4495287A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Process for producing gene products of plasmid having temperature dependent plasmid copy number
Assignee: Takeda Pharma AS
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Filing: 1978-05-22
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Granted: 1985-01-22
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Expires: 1998-05-22
Abstract
Abstract Gene products of plasmid DNA, such as proteins, are prepared in high yields by cultivating bacteria carrying a plasmid which shows a controlled constant plasmid copy number at one temperture and a much higher or totally uncontrolled copy number at a different temperature. The plasmid may be prepared by recombinant DNA technique using a cloning vector showing the temperature dependent plasmid copy number pattern.
Inventors
- 👤 Bernt E. Uhlin
- 👤 Kurt Nordstrom
- 👤 Soeren Molin
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 04:50
CPC Classifications
C; C09; C09K; C09K8/00; C09K8/02; C09K8/04; C09K8/26; C09K23/00; C09K8/28; C09K8/32; C09K8/36; C09K8/42; C09K8/424; C09K8/58; C09K8/584; C09K8/60; C09K8/601; C10; C10L; C10L1/00; C10L1/32; C10L1/328; C12; C12N; C12N15/00; C12N15/01; C12N15/09; C12N15/63; C12N15/67; C12N15/69; Y; Y10; Y10S; Y10S435/00; Y10S435/82
Claims Analysis
Total Claims: 11
Claim 1
Independent Claim
135 words
A method for producing a gene product of DNA, comprising cultivating bacteria carrying a recombinant DNA plasmid showing a controlled constant plasmid copy number when the host bacteria are cultivated at one temperature, and showing, when the host bacteria carrying the plasmid are grown at a different temperature, an altered plasmid copy number control allowing an at least 20 times higher copy number, or a totally uncontrolled copy number under conditions including at least a period of cultivation at or approaching a temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number; said approaching temperature being a temperature at which the host bacteria survive and are still capable of propagation; and harvesting, from the bacterial culture, a gene product of the plasmid.
Claim 2
Independent Claim
135 words
A method for producing a gene product of DNA, comprising cultivating bacteria carrying a recombinant DNA plasmid showing a controlled constant plasmid copy number when the host bacteria are cultivated at one temperature, and showing, when the host bacteria carrying the plasmid are grown at a different temperature, an altered plasmid copy number control allowing an at least 20 times higher copy number, or a totally uncontrolled copy number under conditions including at least a period of cultivation at or approaching a temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number; said approaching temperature being a temperature at which the host bacteria survive and are still capable of propagation; and harvesting, from the bacterial culture, a gene product of the plasmid.
Claim 3
Dependent Claim
76 words
A method as claimed in claim 1 comprising performing cultivation at the temperature at which the plasmid shows a controlled constant copy number during seeding and multiplication stages and until an amount of cells sufficient for production size culture is obtained, and thereafter performing production comprising cultivation at or approaching the temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number, and harvesting.
Claim 4
Dependent Claim
55 words
A method as claimed in claim 1 wherein the cultivation at the temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number is continued until the growth of the bacteria is inhibited by the production of the plasmid or gene products thereof.
Claim 5
Dependent Claim
80 words
A method as claimed in claim 1, wherein the production scale cultivation is performed continuously at a temperature approaching the temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number so as to permit survival of the host cells concomitantly with formation of increased amounts of gene products of the plasmid, and wherein the gene product of the plasmid is continuously or intermittently harvested from the culture.
Claim 6
Dependent Claim
176 words
A method as claimed in claim 1, wherein the period of cultivation at or approaching a temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number is preceded by the propagation of the bacterial culture to reach production size culture, after the propagation the temperature is shifted to a temperature at or substantially at the temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number, and the temperature is maintained for a sufficient period to obtain a substantial amplification of the plasmid, but for a period sufficiently short to avoid any substantial impairment of the bacterial culture, whereafter the temperature is again shifted to a temperature at or approaching the temperature at which the plasmid shows a controlled constant plasmid copy number, and production cultivation of the culture having the amplified plasmid copy number is continued at said temperature at which the plasmid shows a controlled constant plasmid copy number.
Claim 7
Dependent Claim
48 words
A method as claimed in claim 1, wherein the temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number is higher than the temperature at which the plasmid shows a controlled constant plasmid copy number.
Claim 8
Dependent Claim
65 words
A method as claimed in claim 6, wherein the host bacteria are mesophilic bacteria, and wherein the temperature at which the plasmid shows a constant controlled plasmid copy number is up to about 32° C., whereas the temperature at which the plasmid shows an altered copy number control allowing an at least 20 times higher or totally uncontrolled copy number is above about 36° C.
Claim 9
Dependent Claim
184 words
A method as claimed in claim 1, wherein the plasmid is one prepared by combining a cloning vector with a foreign DNA fragment; said plasmid is a plasmid showing a controlled constant plasmid copy number when host bacteria carrying the plasmid are cultivated at one temperature, and showing when the host bacteria carrying the plasmid are grown at a different temperature, an altered plasmid copy number control allowing an at least 20 times higher or totally uncontrolled copy number; said cloning vector carrying for at least one restriction endonuclease, one and only one site susceptible to the endonuclease, said site being such that, after insertion of a fragment of foreign DNA at the site, the resulting recombinant DNA is able to replicate autonomously and with the retention of the capability of showing a controlled constant plasmid copy number when host bacteria carrying the recombinant DNA are cultivated at one temperature, and showing, when the host bacteria carrying the recombinant DNA are growing at a different temperature, an altered plasmid copy number control allowing an at least 20 times higher or totally uncontrolled copy number.
Claim 10
Independent Claim
17 words
A method as claimed in any of claims 1-7, or 8, wherein said plasmid is a mini-plasmid.
Claim 11
Dependent Claim
15 words
A method as claimed in claim 1, wherein the plasmid is a derivative of Rl.
References Cited (Backward Citations)
US4237224A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-01-22 | 1988-07-23 | 1989-01-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-01-22 | 1992-07-23 | 1993-01-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-01-22 | 1996-07-23 | 1997-01-22 | $7700.00 | Window Closed / Lapsed |