US4338397A
Expired / Lapsed
US
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Mature protein synthesis
Assignee: Biogen Inc
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Filing: 1980-04-11
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Granted: 1982-07-06
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Expires: 2000-04-11
Abstract
Abstract A method is provided for synthesizing within a bacterial host, and secreting through the membrane of the host, a selected mature protein or polypeptide. The method involves: (a) cleaving a cloning vehicle to form a cleavage site after a promoter of either (1) a bacterial or phage gene within the cloning vehicle or (2) a DNA fragment of the bacterial or phage gene; (b) forming a hybrid gene by inserting into the cleavage site a non-bacterial DNA fragment which codes for precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide; (c) transforming the host with the cloning vehicle; and then (d) culturing the transformed host to secrete the selected protein or polypeptide. By this method, mature proteins or polypeptides can be produced, free of signal sequences or other chemical substituents, such as an f-met, on the proteins or polypeptides.
Inventors
- 👤 Walter Gilbert
- 👤 Karen Talmadge
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 16:13
CPC Classifications
C; C12; C12N; C12N15/00; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/62; C12N15/09; C12N15/11; C12N15/62; C12N15/625; C12N15/63; C12N15/70; C07K2319/00; C07K2319/01; C07K2319/02
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
139 words
A method of synthesizing within a bacterial host, and secreting through the membrane of the bacterial host, a selected mature protein or polypeptide, which comprises: (a) cleaving a cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host, to form a cleavage site after a promoter of either (1) a bacterial or phage gene within the cloning vehicle or (2) a DNA fragment of the bacterial or phage gene; (b) forming a hybrid gene by inserting into the cleavage site a non-bacterial DNA fragment which codes for a precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide; (c) transforming the bacterial host with the cloning vehicle; and then (d) culturing the transformed bacterial host to secrete the selected protein or polypeptide.
Claim 2
Independent Claim
139 words
A method of synthesizing within a bacterial host, and secreting through the membrane of the bacterial host, a selected mature protein or polypeptide, which comprises: (a) cleaving a cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host, to form a cleavage site after a promoter of either (1) a bacterial or phage gene within the cloning vehicle or (2) a DNA fragment of the bacterial or phage gene; (b) forming a hybrid gene by inserting into the cleavage site a non-bacterial DNA fragment which codes for a precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide; (c) transforming the bacterial host with the cloning vehicle; and then (d) culturing the transformed bacterial host to secrete the selected protein or polypeptide.
Claim 3
Dependent Claim
54 words
The method of claim 1 wherein the bacterial or phage gene or the DNA fragment thereof codes for a normally secreted protein or polypeptide and the cleavage site is before, within or no more than approximately 60 nucleotides after the translational start signal of the bacterial or phage gene or the DNA fragment thereof.
Claim 4
Dependent Claim
54 words
The method of claim 1 wherein the bacterial or phage gene or the DNA fragment thereof codes for a normally non-secreted protein or polypeptide and the cleavage site is before, within or no more than approximately 40 nucleotides after the translational start signal of the bacterial or phage gene or the DNA fragment thereof.
Claim 5
Independent Claim
36 words
The method of claims 2 or 3 wherein the cleavage site is within or no more than approximately 40 nucleotides after the translational start signal of the bacteria or phage gene or the DNA fragment thereof.
Claim 6
Dependent Claim
17 words
The method of claim 1 wherein a DNA fragment of the E. coli penicillinase gene is cleaved.
Claim 7
Dependent Claim
15 words
The method of claim 5 wherein the cloning vehicle is derived from the plasmid pBR322.
Claim 8
Dependent Claim
22 words
The method of claim 1 wherein the non-bacterial DNA fragment codes for preproinsulin, preserum albumin, prehuman growth hormone, preparathyroid hormone, or preinterferon.
Claim 9
Dependent Claim
81 words
A cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in a bacterial host and comprising in order: a promoter of a bacterial or phage gene; a translation start signal; and a non-bacterial DNA fragment which codes for a precursor of a protein or polypeptide, including the signal sequence of the protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame define by the translational start signal.
Claim 10
Dependent Claim
81 words
A cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in a bacterial host and comprising in order: a promoter of a bacterial or phage gene; a translation start signal; and a non-bacterial DNA fragment which codes for a precursor of a protein or polypeptide, including the signal sequence of the protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame define by the translational start signal.
Claim 11
Dependent Claim
21 words
The cloning vehicle of claim 8 which further includes a ribosome binding site between the promoter and the translational start signal.
Claim 12
Dependent Claim
18 words
The cloning vehicle of claim 9 wherein the promoter is the promoter of the E. coli penicillinase gene.
Claim 13
Dependent Claim
32 words
The cloning vehicle of claim 9 which further includes no more than approximately 40 nucleotides of the bacterial or phage gene after the translational start signal and before the non-bacterial DNA fragment.
Claim 14
Dependent Claim
23 words
The cloning vehicle of claim 9 wherein the non-bacterial DNA fragment codes for preproinsulin, preserum albumin, prehuman growth hormone, preparathyroid hormone, or preinterferon.
Claim 15
Dependent Claim
89 words
A bacterial host transformed with a cloning vehicle, the cloning vehicle comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host and comprising in order: a promoter of a bacterial or phage gene; a translational start signal; and a non-bacterial DNA fragment which codes for a precursor of a protein or polypeptide, including the signal sequence of the protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame defined by the translational start signal.
Claim 16
Dependent Claim
89 words
A bacterial host transformed with a cloning vehicle, the cloning vehicle comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host and comprising in order: a promoter of a bacterial or phage gene; a translational start signal; and a non-bacterial DNA fragment which codes for a precursor of a protein or polypeptide, including the signal sequence of the protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame defined by the translational start signal.
Claim 17
Dependent Claim
20 words
The host of claim 13 which further includes a ribosome binding site between the promoter and the translational start signal.
Claim 18
Dependent Claim
22 words
The host of claim 14 wherein the non-bacterial DNA fragment codes for preproinsulin, preserum albumin, prehuman growth hormone, preparathyroid hormone, or preinterferon.
Claim 19
Independent Claim
118 words
A method of synthesizing within a bacterial host, and secreting through the membrane of the bacterial host, a selected mature protein or polypeptide, which comprises culturing the bacterial host; the bacterial host being transformed with a cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host and comprising in order: a promoter of a bacterial or phage gene; a translational start signal; and a non-bacterial DNA fragment which codes for a precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame defined by the translational start signal.
Claim 20
Independent Claim
118 words
A method of synthesizing within a bacterial host, and secreting through the membrane of the bacterial host, a selected mature protein or polypeptide, which comprises culturing the bacterial host; the bacterial host being transformed with a cloning vehicle, comprising a plasmid, phage DNA or other DNA sequence which is able to replicate in the bacterial host and comprising in order: a promoter of a bacterial or phage gene; a translational start signal; and a non-bacterial DNA fragment which codes for a precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide; the reading frame of the non-bacterial DNA fragment being located in the reading frame defined by the translational start signal.
Claim 21
Dependent Claim
23 words
The method of claim 16 wherein the cloning vehicle further includes a ribosome binding site between the promoter and the translational start signal.
Claim 22
Dependent Claim
17 words
The method of claim 17 wherein the promoter is the promoter of the E. coli penicillinase gene.
Claim 23
Dependent Claim
34 words
The method of claim 17 wherein the cloning vehicle further includes no more than approximately 40 nucleotides of the bacterial or phage gene after the translational start signal and before the non-bacterial DNA fragment.
References Cited (Backward Citations)
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US4237224A
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GB1516458A
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GB1565190A
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GB1568047A
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1985-07-05 | 1986-01-04 | 1986-07-06 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1989-07-05 | 1990-01-04 | 1990-07-06 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1993-07-05 | 1994-01-04 | 1994-07-06 | $7700.00 | Window Closed / Lapsed |