US4767708A
Expired / Lapsed
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Enzyme amplification and purification
Assignee: Carnegie Mellon University
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Filing: 1984-08-07
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Granted: 1988-08-30
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Expires: 2004-08-07
Abstract
Abstract Restriction enzymes are used to remove from DNA a complete and undamaged structural gene coding region for the expression of DNA polymerase I (polA) without the gene's natural promoter or with only a significantly damaged portion of the gene's natural promoter. Also by the use of restriction enzymes, a segment from a plasmid cloning vector is excised at a position adjacent to a promoter which is conditionally controllable and may be more powerful than the damaged or removed promoter. The gene for DNA polymerase I is enzymatically cloned into said vector at the position of said removed segment and adjacent to said conditionally controllable promoter. Multicopies of the cloned vector are introduced into a host baterial strain (E. coli). The host strain is then cultured so that the cell colony grows and replicates new generations containing replicated foreign plasmid. During such said replication the activity of said controllable promoter is repressed. After the cell colony has grown, the repression of said controllable promoter is removed and the cells express an amplified amount of DNA polymerase I which is lethal or inhibitory to the cells. An improved procedure is disclosed comprising a sequence of steps for harvesting purified DNA polymerase I.
Inventors
- 👤 Edwin G. Minkley, Jr.
- 👤 William E. Brown
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 00:39
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252
Claims Analysis
Total Claims: 50
Claim 1
Independent Claim
59 words
A recombinant plasmid containing a cloned complete structural gene coding region isolated from a bacterial source for the expression of DNA polymerase I, under operable control of a conditionally controllable foreign promoter functionally linked to said structural gene coding region, said foreign promoter being functional to express said DNA polymerase I in a suitable bacterial or yeast host system.
Claim 2
Dependent Claim
59 words
A recombinant plasmid containing a cloned complete structural gene coding region isolated from a bacterial source for the expression of DNA polymerase I, under operable control of a conditionally controllable foreign promoter functionally linked to said structural gene coding region, said foreign promoter being functional to express said DNA polymerase I in a suitable bacterial or yeast host system.
Claim 3
Dependent Claim
20 words
The recombinant plasmid of claim 1 wherein said foreign promoter is more active than the natural promoter of said gene.
Claim 4
Dependent Claim
15 words
The recombinant plasmid of claim 1 wherein said foreign promoter is a negatively regulated promoter.
Claim 5
Dependent Claim
19 words
The recombinant plasmid of claim 3 wherein said negatively regulated promoter is the lac promoter or the trp promoter.
Claim 6
Dependent Claim
18 words
The recombinant plasmid of claim 3 wherein said negatively regulated promoter is the leftward promoter of phage lambda.
Claim 7
Dependent Claim
15 words
The recombinant plasmid of claim 1 wherein said foreign promoter is a positively regulated promoter.
Claim 8
Dependent Claim
15 words
The recombinant plasmid of claim 6 wherein said positively regulated promoter is the mal promoter.
Claim 9
Dependent Claim
22 words
The recombinant plasmid of claim 6 wherein said positively regulated promoter is one of the late promoters of bacteriophage T 7 .
Claim 10
Dependent Claim
11 words
The recombinant plasmid of claim 1 within a yeast host strain.
Claim 11
Dependent Claim
11 words
The recombinant plasmid of claim 1 within a bacterial host strain.
Claim 12
Dependent Claim
12 words
The recombinant plasmid of claim 1 within an E. coli host strain.
Claim 13
Dependent Claim
17 words
The recombinant plasmid of claim 3 within a host strain with a repressor for said foreign promoter.
Claim 14
Dependent Claim
17 words
The recombinant plasmid of claim 6 within a host strain with an activator for said foreign promoter.
Claim 15
Dependent Claim
10 words
The recombinant plasmid of claim 1 within a host strain.
Claim 16
Dependent Claim
22 words
The host strain of claim 14 wherein said expression of said DNA polymerase I by said foreign promoter has not been activated.
Claim 17
Dependent Claim
18 words
The host strain of claim 14 wherein said expression of said DNA polymerase I by said foreign promoter.
Claim 18
Dependent Claim
13 words
The recombinant plasmid of claim 12 wherein said host strain is E. coli.
Claim 19
Dependent Claim
13 words
The recombinant plasmid of claim 13 wherein said host strain is E. coli.
Claim 20
Dependent Claim
13 words
The recombinant plasmid of claim 14 wherein said host strain is E. coli.
Claim 21
Dependent Claim
4 words
The recombinant plasmid pMP5.
Claim 22
Dependent Claim
4 words
The recombinant plasmid pMP5.
Claim 23
Dependent Claim
5 words
E. coli strain ATL 100.
Claim 24
Dependent Claim
5 words
E. coli strain ATL 100.
Claim 25
Dependent Claim
17 words
The recombinant plasmid of claim 1 without any portion of the natural promoter of said structural gene.
Claim 26
Dependent Claim
27 words
The recombinant plasmid of claim 1 with the natural promoter of said structural gene mutated to be less active than said natural promoter in its wild-type state.
Claim 27
Dependent Claim
29 words
The recombinant plasmid of claim 1 in a host strain wherein the natural promoter of said structural gene is less active than said natural promoter in its wild-type state.
Claim 28
Dependent Claim
104 words
A process for constructing a recombinant plasmid for the expression of DNA polymerase I, said DNA polymerase I including large and small fragments thereof, comprising the steps of: excising enzymatically from a DNA molecule the complete structural gene coding region isolated from a bacterial source of said DNA polymerase I; cloning said complete structural gene coding region into a vector plasmid, said complete structural gene coding region under the operable control of a conditionally controllable foreign promoter functionally linked to said complete structural gene coding region, said foreign promoter functional in a suitable bacterial or yeast host system, said cloning forming a recombinant plasmid.
Claim 29
Dependent Claim
104 words
A process for constructing a recombinant plasmid for the expression of DNA polymerase I, said DNA polymerase I including large and small fragments thereof, comprising the steps of: excising enzymatically from a DNA molecule the complete structural gene coding region isolated from a bacterial source of said DNA polymerase I; cloning said complete structural gene coding region into a vector plasmid, said complete structural gene coding region under the operable control of a conditionally controllable foreign promoter functionally linked to said complete structural gene coding region, said foreign promoter functional in a suitable bacterial or yeast host system, said cloning forming a recombinant plasmid.
Claim 30
Dependent Claim
27 words
The process of claim 25 wherein the natural promoter of said structural gene is mutated to be less active than said natural promoter in its wild-type state.
Claim 31
Dependent Claim
24 words
The process of claim 25 wherein said vector plasmid promoter or foreign promoter is more active than the natural promoter of said structural gene.
Claim 32
Dependent Claim
21 words
The process of claim 25 including the step of transforming said recombinant plasmid into a suitable bacterial or yeast host strain.
Claim 33
Dependent Claim
12 words
The process of claim 28 wherein said host strain is E. coli.
Claim 34
Dependent Claim
51 words
The process of claim 28 including the step of culturing cells of said host strain containing said plasmid in a nutrient broth to form a desired amount of viable cells containing replications of said recombinant plasmid, said culturing occurring in the absence of activation of the expression of DNA polymerase I.
Claim 35
Dependent Claim
22 words
The process of claim 30 wherein the temperature at which said culturing occurs fails to activate the expression of DNA polymerase I.
Claim 36
Dependent Claim
21 words
The process of claim 30 wherein said culturing occurs under environmental control which does not activate expression of DNA polymerase I.
Claim 37
Dependent Claim
20 words
The process of claim 30 wherein said culturing occurs under environmental control which represses the expression of DNA polymerase I.
Claim 38
Dependent Claim
44 words
The process of claim 30 including the additional step of terminating repression or inducing activation of expression of DNA polymerase I so that said desired amount or viable cells expresses an amount of DNA polymerase I greater than the wild-type amount in said cells.
Claim 39
Dependent Claim
62 words
The process of claim 34 wherein said controllable promoter is the bacteriophage lambda leftward promoter (p L ) subject to control by a temperature sensitive cI repressor gene and said culturing step occurs at a temperature in the range of 15° to 34° C. and said DNA polymerase I expression step occurs at a temperature in the range 35° to 45° C.
Claim 40
Dependent Claim
19 words
The process of claim 34 including the step of isolating said expressed DNA polymerase I from said nutrient broth.
Claim 41
Dependent Claim
13 words
The desired amount of viable cells produced in the process of claim 30.
Claim 42
Dependent Claim
12 words
The desired amount of cells produoed by the process of claim 34.
Claim 43
Dependent Claim
26 words
A host strain of cells containing a foreign plasmid capable of expressing DNA polymerase I to a level above the wild-type level in said host strain.
Claim 44
Dependent Claim
26 words
A host strain of cells containing a foreign plasmid capable of expressing DNA polymerase I to a level above the wild-type level in said host strain.
Claim 45
Dependent Claim
30 words
A process comprising culturing said host strain of cells of claim 39 in a nutrient broth to express said DNA polymerase I above said wild-type level in said host strain.
Claim 46
Dependent Claim
16 words
The process of claim 1 wherein said DNA polymerase I is E. coli DNA polymerase I.
Claim 47
Dependent Claim
16 words
The process of claim 26 wherein said DNA polymerase I is E. coli DNA polymerase I.
Claim 48
Dependent Claim
43 words
The recombinant plasmid of claim 23, wherein said mutation is accomplished by using a restriction enzyme, wherein said restriction enzyme cutting within said natural promoter, mutating said natural promoter and eliminating or greatly reducing unregulated expression of the activity of said natural promoter.
Claim 49
Dependent Claim
91 words
The recombinant plasmid of claim 23, wherein said mutation is accomplished by the process comprising the steps of: cutting a DNA molecule containing said structural gene with a restriction enzyme, said cutting of said structural gene occurring distal to said complete structural gene coding region; and digesting said cut DNA molecule with a nuclease, said nuclease carrying out a step-wise removal of nucleotides of said cut DNA molecule to a location within said natural promoter, said step-wise removal eliminating or greatly reducing unregulated expression of the activity of said natural promoter.
Claim 50
Dependent Claim
49 words
The recombinant plasmid of claim 23, wherein said mutation is accomplished by using a restriction enzyme, said restriction enzyme cutting between said natural promoter and said complete structural gene coding region of said DNA polymerase I, eliminating or greatly reducing unregulated expression of the activity of said natural promoter.
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) | 1991-08-30 | 1992-02-29 | 1992-08-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-08-30 | 1996-02-29 | 1996-08-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-08-30 | 2000-02-29 | 2000-08-30 | $7700.00 | Window Closed / Lapsed |