Patent US4757013A successfully refreshed from Google Patents.
US4757013A
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Cloning vehicles for polypeptide expression in microbial hosts
Assignee: Research Foundation of the State University of New York
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Filing: 1986-06-26
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Granted: 1988-07-12
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Expires: 2006-06-26
Abstract
Abstract Methods and compositions are provided for regulated expression and secretion of polypeptides in transformed bacterial hosts. A novel class of plasmid cloning vehicles includes a DNA sequence coding for the desired polypeptide (or an insertion site therefor) linked for transcriptional expression in reading phase with four functional fragments derived from the lipoprotein gene of E. coli. The plasmids also include a DNA fragment coding for the signal peptide of the ompA protein of E. coli, positioned such that the desired polypeptide is expressed with the ompA signal peptide at its amino terminus, thereby allowing efficient secretion across the cytoplasmic membrane. The plasmids further include a DNA sequence coding for a specific segment of the E. coli lac promoter-operator, which is positioned in the proper orientation for transcriptional expression of the desired polypeptide, as well as a separate functional E. coli lacI gene coding for the associated repressor molecule which can interact with the lac promoter-operator to prevent transcription therefrom. Expression of the desired polypeptide is under the control of both the lipoprotein promoter and the lac promoter-operator, although transcription from either promoter is normally blocked by the repressor molecule. However, the repressor can be selectively inactivated by means of an inducer molecule to permit transcriptional expression of the desired polypeptide from both promoters. The methods utilize such plasmids to introduce genetic capability into micro-organisms for the production of normally secreted proteins, such as medically or commercially useful hormones, enzymes, immunogenic proteins, or intermediates therefor, but only in the presence of an appropriate inducer.
Inventors
- 👤 Masayori Inouye
- 👤 Yoshihiro Masui
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 18:29
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/70; C12N15/11; C12N15/62; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/12
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
139 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 the promoter of the lipoprotein gene of Escherichia coli, linked in reading phase with (a) a second DNA sequence located downstream of said lipoprotein promoter and coding for the segment between positions -40 and +65 of the β-galactosidase promoter-operator of Escherichia coli, (b) a third DNA sequence located downstream of said second DNA sequence and coding for the signal peptide of the ompA gene of Escherichia coli, (c) a fourth DNA sequence located downstream of said third DNA sequence and coding for the amino acid sequence of said at least one polypeptide, said plasmid also comprising a fifth DNA sequence coding for the lacI gene of Escherichia coli.
Claim 2
Dependent Claim
139 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 the promoter of the lipoprotein gene of Escherichia coli, linked in reading phase with (a) a second DNA sequence located downstream of said lipoprotein promoter and coding for the segment between positions -40 and +65 of the β-galactosidase promoter-operator of Escherichia coli, (b) a third DNA sequence located downstream of said second DNA sequence and coding for the signal peptide of the ompA gene of Escherichia coli, (c) a fourth DNA sequence located downstream of said third DNA sequence and coding for the amino acid sequence of said at least one polypeptide, said plasmid also comprising a fifth DNA sequence coding for the lacI gene of Escherichia coli.
Claim 3
Dependent Claim
41 words
A plasmid in accordance with claim 1 further comprising a sixth DNA sequence located downstream of said lipoprotein promoter and upstream of said second DNA sequence and coding for at least a portion of the 5'-untranslated region of said lipoprotein gene.
Claim 4
Dependent Claim
59 words
A plasmid in accordance with claim 2 further comprising a seventh DNA sequence located downstream of said fourth DNA sequence and comprising the 3'-untranslated region and the transcription termination signal of said lipoprotein gene, wherein said fourth DNA sequence is located upstream of said 3'-untranslated region and wherein said 3'-untranslated region is located upstream of said transcription termination signal.
Claim 5
Dependent Claim
61 words
A plasmid in accordance with claim 3 wherein said seventh DNA sequence further comprises a terminal portion of the structural sequence of said lipoprotein gene, said terminal portion coding for the DNA segment commencing with position +168 of said lipoprotein structural gene, and wherein said terminal portion is located upstream of said 3'-untranslated region and downstream of said fourth DNA sequence.
Claim 6
Dependent Claim
19 words
A plasmid in accordance with claim 4 wherein said at least one polypeptide comprises the β-lactamase of Escherichia coli.
Claim 7
Dependent Claim
17 words
A plasmid in accordance with claim 4 wherein said at least one polypeptide comprises a mammalian hormone.
Claim 8
Dependent Claim
15 words
A plasmid in accordance with claim 6 wherein said mammalian hormone is human growth hormone.
Claim 9
Dependent Claim
224 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 comprising DNA coding for the promoter of the lipoprotein gene of Escherichia coli and at least a portion of the 5'-untranslated region of the lipoprotein gene of Escherichia coli, said portion of said 5'-untranslated region located downstream of said promoter, said first DNA sequence linked with (a) a second DNA sequence located downstream of said first DNA sequence and coding for the segment between positions -40 and +65 of the β-galactosidase promoter-operator of Escherichia coli, (b) a third DNA sequence located downstream of said second DNA sequence and coding for the signal peptide of the ompA gene of Escherichia coli, (c) a fourth DNA sequence located downstream of said third DNA sequence and coding for a translation initiation codon adjacent the recognition sequence of at least one restriction endonuclease for incorporation in said plasmid of a fifth DNA sequence coding for the amino acid sequence of said at least one polypeptide, and (d) a sixth DNA sequence located downstream of said fifth DNA sequence and coding for the 3'-untranslated region and the transcription termination signal of said lipoprotein gene, said plasmid also comprising a seventh DNA sequence coding for the lacI gene of Escherichia coli.
Claim 10
Dependent Claim
224 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 comprising DNA coding for the promoter of the lipoprotein gene of Escherichia coli and at least a portion of the 5'-untranslated region of the lipoprotein gene of Escherichia coli, said portion of said 5'-untranslated region located downstream of said promoter, said first DNA sequence linked with (a) a second DNA sequence located downstream of said first DNA sequence and coding for the segment between positions -40 and +65 of the β-galactosidase promoter-operator of Escherichia coli, (b) a third DNA sequence located downstream of said second DNA sequence and coding for the signal peptide of the ompA gene of Escherichia coli, (c) a fourth DNA sequence located downstream of said third DNA sequence and coding for a translation initiation codon adjacent the recognition sequence of at least one restriction endonuclease for incorporation in said plasmid of a fifth DNA sequence coding for the amino acid sequence of said at least one polypeptide, and (d) a sixth DNA sequence located downstream of said fifth DNA sequence and coding for the 3'-untranslated region and the transcription termination signal of said lipoprotein gene, said plasmid also comprising a seventh DNA sequence coding for the lacI gene of Escherichia coli.
Claim 11
Dependent Claim
61 words
A plasmid in accordance with claim 8 wherein said sixth DNA sequence further comprises a terminal portion of the structural sequence of said lipoprotein gene, said terminal portion coding for the DNA segment commencing with position +168 of said lipoprotein structural gene, and wherein said terminal portion is located upstream of said 3'-untranslated region and downstream of said fourth DNA sequence.
Claim 12
Dependent Claim
27 words
A plasmid in accordance with claim 9 wherein said fourth DNA sequence comprises DNA sequences recognized by the Eco RI, Hind III and Bam HI restriction endonucleases.
Claim 13
Dependent Claim
14 words
A plasmid which is selected from the group consisting of pIN-III-ompA1, pIN-III-ompA2 and pIN-III-ompA3.
Claim 14
Dependent Claim
14 words
A plasmid which is selected from the group consisting of pIN-III-ompA1, pIN-III-ompA2 and pIN-III-ompA3.
Claim 15
Independent Claim
270 words
A method for producing a polypeptide in a transformed bacterial host comprising the steps of (a) selecting a recombinant plasmid comprising a first DNA sequence comprising DNA coding for the promoter of the lipoprotein gene of Escherichia coli and at least a portion of the 5'-untranslated region of the lipoprotein gene of Escherichia coli, said portion of said 5'-untranslated region located downstream of said promoter, said first DNA sequence linked with (i) a second DNA sequence located downstream of said first DNA sequence and coding for the segment between positions -40 and +65 of the β-galactosidase promoter-operator of Escherichia coli, (ii) a third DNA sequence located downstream of said second DNA sequence and coding for the signal peptide of the ompA gene of Escherichia coli, (iii) a fourth DNA sequence located downstream of said third DNA sequence and coding for a translation initiation codon adjacent the recognition sequence of at least one restriction endonuclease for incorporation in said plasmid of a fifth DNA sequence coding for the amino acid sequence of said polypeptide, and (iv) a sixth DNA sequence located downstream of said fifth DNA sequence and coding for the 3'-untranslated region and the transcription termination signal of said lipoprotein gene, said plasmid also comprising a seventh DNA sequence coding for the lacI gene of Escherichia coli, (b) inserting said fifth DNA sequence within said fourth DNA sequence, (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, (e) adding to said population a lactose inducer, and (f) producing said polypeptide from said population.
Claim 16
Independent Claim
270 words
A method for producing a polypeptide in a transformed bacterial host comprising the steps of (a) selecting a recombinant plasmid comprising a first DNA sequence comprising DNA coding for the promoter of the lipoprotein gene of Escherichia coli and at least a portion of the 5'-untranslated region of the lipoprotein gene of Escherichia coli, said portion of said 5'-untranslated region located downstream of said promoter, said first DNA sequence linked with (i) a second DNA sequence located downstream of said first DNA sequence and coding for the segment between positions -40 and +65 of the β-galactosidase promoter-operator of Escherichia coli, (ii) a third DNA sequence located downstream of said second DNA sequence and coding for the signal peptide of the ompA gene of Escherichia coli, (iii) a fourth DNA sequence located downstream of said third DNA sequence and coding for a translation initiation codon adjacent the recognition sequence of at least one restriction endonuclease for incorporation in said plasmid of a fifth DNA sequence coding for the amino acid sequence of said polypeptide, and (iv) a sixth DNA sequence located downstream of said fifth DNA sequence and coding for the 3'-untranslated region and the transcription termination signal of said lipoprotein gene, said plasmid also comprising a seventh DNA sequence coding for the lacI gene of Escherichia coli, (b) inserting said fifth DNA sequence within said fourth DNA sequence, (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, (e) adding to said population a lactose inducer, and (f) producing said polypeptide from said population.
Claim 17
Dependent Claim
233 words
A bacterial transformant comprising a plasmid comprised of a first DNA sequence comprising DNA coding for the promoter of the lipoprotein gene of Escherichia coli and at least a portion of the 5'-untranslated region of the lipoprotein gene of Escherichia coli, said portion of said 5'-untranslated region located downstream of said promoter, said first DNA sequence linked with (a) a second DNA sequence located downstream of said first DNA sequence and coding for the segment between positions -40 and +65 of the β-galactosidase promoter-operator of Escherichia coli, (b) a third DNA sequence located downstream of said second DNA sequence and coding for the signal peptide of the ompA gene of Escherichia coli, (c) a fourth DNA sequence located downstream of said third DNA sequence coding for a translation initiation codon adjacent the recognition sequence of at least one restriction endonuclease for incorporation in said plasmid of a fifth DNA sequence coding for the amino acid sequence of a polypeptide, and (d) a sixth DNA sequence located downstream of said fifth DNA sequence and coding for the 3'-untranslated region and the transcription termination signal of said lipoprotein gene, said plasmid also comprising a seventh DNA sequence coding for the lacI gene of Escherichia coli, said transformant being capable of producing the polypeptide upon fermentation in an aqueous nutrient medium containing assimilable sources fo carbon, nitrogen and inorganic substances, and also containing a lactose inducer.
Claim 18
Dependent Claim
233 words
A bacterial transformant comprising a plasmid comprised of a first DNA sequence comprising DNA coding for the promoter of the lipoprotein gene of Escherichia coli and at least a portion of the 5'-untranslated region of the lipoprotein gene of Escherichia coli, said portion of said 5'-untranslated region located downstream of said promoter, said first DNA sequence linked with (a) a second DNA sequence located downstream of said first DNA sequence and coding for the segment between positions -40 and +65 of the β-galactosidase promoter-operator of Escherichia coli, (b) a third DNA sequence located downstream of said second DNA sequence and coding for the signal peptide of the ompA gene of Escherichia coli, (c) a fourth DNA sequence located downstream of said third DNA sequence coding for a translation initiation codon adjacent the recognition sequence of at least one restriction endonuclease for incorporation in said plasmid of a fifth DNA sequence coding for the amino acid sequence of a polypeptide, and (d) a sixth DNA sequence located downstream of said fifth DNA sequence and coding for the 3'-untranslated region and the transcription termination signal of said lipoprotein gene, said plasmid also comprising a seventh DNA sequence coding for the lacI gene of Escherichia coli, said transformant being capable of producing the polypeptide upon fermentation in an aqueous nutrient medium containing assimilable sources fo carbon, nitrogen and inorganic substances, and also containing a lactose inducer.
Claim 19
Dependent Claim
15 words
The transformant of claim 13 wherein the transformant is selected from the species Escherichia coli.
Claim 20
Dependent Claim
19 words
The transformant of claim 14 wherein said plasmid is selected from the group consisting of pINIII-ompA1, pINIII-ompA2 and pINIII-ompA3.
References Cited (Backward Citations)
No citation records indexed.
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