EP0055942A2
Expired / Lapsed
EP
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Plasmid cloning vehicles
Assignee: Research Foundation of the State University of New York
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Filing: 1981-12-31
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Granted: —
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Expires: 2001-12-31
Abstract
Abstract Methods and compositions are provided for expression of polypeptides in transformed bacterial hosts. A novel class of plasmid cloning vehicles includes a DNA sequence coding for the desired polypeptide linked in reading phase with one or more functional fragments derived from an outer membrane protein gene of a gram-negative bacterium. The methods utilize such plasmids to introduce genetic capability into micro-organisms for the production of proteins, such as medically or commercially useful hormones, enzymes, immunogenic proteins, or intermediates therefor.
Inventors
- 👤 Masayori Inouye
- 👤 Kenzo Nakamura
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 20:45
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/62; C07K2319/00; C07K2319/01; C07K2319/02
Claims Analysis
Total Claims: 41
Claim 1
Independent Claim
80 words
A recombinant plasmid suited.for use as a cloning vehicle for expression of at least one polypeptide in a transformed bacterial host, said plasmid comprising a first DNA sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium linked in reading phase with a . second- DNA sequence coding for the amino acid sequence of said at least one polypeptide, or with an insertion site adapted to receive said second DNA sequence.
Claim 2
Dependent Claim
80 words
A recombinant plasmid suited.for use as a cloning vehicle for expression of at least one polypeptide in a transformed bacterial host, said plasmid comprising a first DNA sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium linked in reading phase with a . second- DNA sequence coding for the amino acid sequence of said at least one polypeptide, or with an insertion site adapted to receive said second DNA sequence.
Claim 3
Dependent Claim
66 words
A recombinant plasmid suited for use as a cloning vehicle for expression of at least one polypeptide in a transformed bacterial host, said plasmid comprising a DNA sequence coding for the amino acid sequence of said at least one polypeptide linked in reading phase with a DNA sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium.
Claim 4
Dependent Claim
66 words
A recombinant plasmid suited for use as a cloning vehicle for expression of at least one polypeptide in a transformed bacterial host, said plasmid comprising a DNA sequence coding for the amino acid sequence of said at least one polypeptide linked in reading phase with a DNA sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium.
Claim 5
Dependent Claim
74 words
A recombinant plasmid suited for use as a cloning vehicle for expression of at least one polypeptide in a transformed bacterial host, said plasmid comprising a first DNA sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium linked in reading phase with an insertion site adopted to receive a second DNA sequence coding for the amino acid sequence of said at least one polypeptide.
Claim 6
Dependent Claim
74 words
A recombinant plasmid suited for use as a cloning vehicle for expression of at least one polypeptide in a transformed bacterial host, said plasmid comprising a first DNA sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium linked in reading phase with an insertion site adopted to receive a second DNA sequence coding for the amino acid sequence of said at least one polypeptide.
Claim 7
Dependent Claim
19 words
The plasmid of claim 1, 2 or 3 wherein said at least one functional fragment comprises a promoter signal.
Claim 8
Dependent Claim
18 words
The plasmid of claim 4 wherein said at least one functional fragment also comprises a 5'- untranslated region.
Claim 9
Dependent Claim
26 words
The plasmid of any one of claims 1 to 5 wherein said at least one functional fragment comprises a 3'-untranslated region and a transcription termination signal.
Claim 10
Dependent Claim
39 words
The plasmid of any one of claims 1 to 6 wherein the insertion site lies downstream of the promoter signal and downstream of the 5'-untranslated region and upstream of the 3'-untranslated region and upstream of the transcription termination signal.
Claim 11
Dependent Claim
17 words
The plasmid of any one of claims 1 to 7 wherein said gram-negative bacterium is Escherichia coli.
Claim 12
Dependent Claim
29 words
The plasmid of claim 8 wherein said outer membrane protein gene comprises the lipoprotein gene of Escherichia coli, and said outer membrance protein comprises the lipoprotein of Escherichia coli.
Claim 13
Dependent Claim
20 words
The plasmid of any one of claims 1 to 9 wherein said at least one polypeptide comprises a mammalian hormone.
Claim 14
Dependent Claim
42 words
The plasmid of claim 3 or any one of claims 7 to 10 wherein the insertion site lies downstream of the promoter signal and downstream of the 5'-untranslated region and upstream of the 3'-untranslated region and upstream of the transcription termination signal.
Claim 15
Dependent Claim
31 words
The plasmid of claim 11 wherein said first DNA sequence further codes for a peptide extension capable of directing translocation of a product across the cytoplasmic membrane of the bacterial host.
Claim 16
Dependent Claim
19 words
The plasmid of claim 11 wherein the insertion site lies within the DNA sequence coding for the peptide extension.
Claim 17
Dependent Claim
23 words
The plasmid of claim 11 wherein the insertion site lies at the 3' terminus of the DNA sequence coding for the peptide extension.
Claim 18
Dependent Claim
29 words
The plasmid of claim 11 wherein said first DNA sequence further codes for at most the entire amino acid sequence of an outer membrane protein of a gram-negative bacterium.
Claim 19
Dependent Claim
32 words
The plasmid of claim 15 wherein the insertion site lies within the DNA sequence coding for at most the entire amino acid sequence of an outer membrane protein of a gram-negative bacterium.
Claim 20
Dependent Claim
30 words
The plasmid of claims 3 and 9 wherein the insertion site comprises a DNA sequence containing the recognition sequences for the Eco RI, Hind III and Bam HI restriction endonucleases.
Claim 21
Independent Claim
93 words
A method for producing a polypeptide in a transformed bacterial host comprising the steps of (a) creating a recombinant plasmid including a DNA sequence coding for the amino acid sequence of said polypeptide linked in reading phase with a DNA sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium, (b) inserting said plasmid in said bacterial host by transformation, (c) isolating and culturing said bacterial host to produce a large population of said bacterial host, and (d) producing said polypeptide from said population.
Claim 22
Independent Claim
93 words
A method for producing a polypeptide in a transformed bacterial host comprising the steps of (a) creating a recombinant plasmid including a DNA sequence coding for the amino acid sequence of said polypeptide linked in reading phase with a DNA sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium, (b) inserting said plasmid in said bacterial host by transformation, (c) isolating and culturing said bacterial host to produce a large population of said bacterial host, and (d) producing said polypeptide from said population.
Claim 23
Independent Claim
114 words
A method for producing a polypeptide in a transformed bacterial host comprising the steps of (a) creating a recombinant plasmid including a first DNA 'sequence coding for at least one functional fragment derived from an outer membrance protein gene of a gram-negative bacterium linked in reading phase with an insertion site adapted to receive a second DNA sequence coding for the amino acid sequence of said polypeptide, (b) inserting said second DNA sequence within the plasmid at the insertion site, (c) inserting the plasmid in said bacterial host by transformation, (d) isolating and culturing said bacterial host to produce a large population of said bacterial host, and (e) producing said polypeptide from said population.
Claim 24
Independent Claim
114 words
A method for producing a polypeptide in a transformed bacterial host comprising the steps of (a) creating a recombinant plasmid including a first DNA 'sequence coding for at least one functional fragment derived from an outer membrance protein gene of a gram-negative bacterium linked in reading phase with an insertion site adapted to receive a second DNA sequence coding for the amino acid sequence of said polypeptide, (b) inserting said second DNA sequence within the plasmid at the insertion site, (c) inserting the plasmid in said bacterial host by transformation, (d) isolating and culturing said bacterial host to produce a large population of said bacterial host, and (e) producing said polypeptide from said population.
Claim 25
Independent Claim
18 words
The method of claims 18 or 19 wherein said at least one functional fragment comprises a promoter signal.
Claim 26
Dependent Claim
20 words
The method of claim 20 wherein said at least one functional fragment comprises a promoter signal and a 5'-untranslated region.
Claim 27
Dependent Claim
23 words
The method of claim 18 or 19 wherein said at least one functional fragment comprises a 3'-untranslated region and a transcription termination signal.
Claim 28
Independent Claim
33 words
The method of any one of claims 18 to 22 wherein said at least one functional fragment comprises a promoter signal, a 5'-untranslated region, a 3'- untranslated region and a transcription termination signal.
Claim 29
Independent Claim
17 words
The method of any one of claims 18 to 23 -wherein said gram-negative bacterium is Escherichia coli.
Claim 30
Independent Claim
33 words
The method of-any one of claims 18 to 24 wherein said outer membrane protein gene comprises the lipoprotein gene of Escherichia coli, and said outer membrane protein comprises the lipoprotein of Escherichia coli.
Claim 31
Independent Claim
17 words
The method of any one of claims 18 to 25 wherein said polypeptide is a mammalian hormone.
Claim 32
Dependent Claim
11 words
The method of claim 26 wherein said polypeptide is human proinsulin.
Claim 33
Independent Claim
39 words
The method of any one of claims 19 to 27 wherein the insertion site lies downstream of the promoter signal and downstream of the 5'-untranslated region and upstream of the 3'-untranslated region and upstream of the transcription termination signal.
Claim 34
Dependent Claim
31 words
The method of claim 28 wherein said first DNA sequence further codes for a peptide extension capable of directing translocation of a product across the cytoplasmic membrane of the bacterial host.
Claim 35
Dependent Claim
19 words
The method of claim 29 wherein the insertion site lies within the DNA sequence coding for the peptide extension.
Claim 36
Dependent Claim
23 words
The method of claim 28 wherein the insertion site lies at the 3' terminus of the DNA sequence coding for the peptide extension.
Claim 37
Dependent Claim
30 words
The method of claim 28 wherein said first D NA sequence further codes for at most the entire amino acid sequence of an outer membrane protein of a gram-negative bacterium.
Claim 38
Dependent Claim
32 words
The method of claim 32 wherein the insertion site lies within the DNA sequence coding for- at most the entire amino acid sequence of an outer membrane protein of a gram-negative bacterium.
Claim 39
Independent Claim
33 words
The method of any one of claims 19 to 33 wherein the insertion site comprises a DNA sequence containing the recognition sequences for the Eco RI, Hind III and Bam HI restriction endonucleases.
Claim 40
Dependent Claim
62 words
A micro-organism derived from Escherichia coli, containing a plasmid comprised of a DNA sequence coding for the amino acid sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium, said culture being capable of producing the polypeptide upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.
Claim 41
Dependent Claim
62 words
A micro-organism derived from Escherichia coli, containing a plasmid comprised of a DNA sequence coding for the amino acid sequence coding for at least one functional fragment derived from an outer membrane protein gene of a gram-negative bacterium, said culture being capable of producing the polypeptide upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.
References Cited (Backward Citations)
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