US4624926A
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Novel cloning vehicles for polypeptide expression in microbial hosts
Assignee: Research Foundation of the State University of New York
•
Filing: 1982-03-03
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Granted: 1986-11-25
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Expires: 2002-03-03
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 gramnegative 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
US
Data Source
google_patents
Last Synchronized
2026-10-29 07:48
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/62; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C07K2319/00; C07K2319/01; C07K2319/02; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/30
Claims Analysis
Total Claims: 71
Claim 1
Independent Claim
112 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid comprising a first DNA sequence coding for the amino acid sequence of said at least one polypeptide linked in reading phase with a second DNA sequence coding for at least one functional fragment derived from an outer membrane lipoprotein gene of Escherichia coli and selected from the group consisting of (a) the promoter, (b) the 5'-untranslated region, (c) the 3'-untranslated region and (d) the transcription termination signal of said lipoprotein gene, said second DNA sequence including at least the promoter of said lipoprotein gene.
Claim 2
Dependent Claim
112 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid comprising a first DNA sequence coding for the amino acid sequence of said at least one polypeptide linked in reading phase with a second DNA sequence coding for at least one functional fragment derived from an outer membrane lipoprotein gene of Escherichia coli and selected from the group consisting of (a) the promoter, (b) the 5'-untranslated region, (c) the 3'-untranslated region and (d) the transcription termination signal of said lipoprotein gene, said second DNA sequence including at least the promoter of said lipoprotein gene.
Claim 3
Dependent Claim
153 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid comprising a first DNA sequence coding for at least one functional fragment derived from an outer membrane lipoprotein gene of Escherichia coli and selected from the group consisting of (a) the promoter, (b) the 5'-untranslated region, (c) the 3'-untranslated region and (d) the transcription termination signal of said lipoprotein gene, said first DNA sequence including at least the promoter of said lipoprotein gene, said plasmid further comprising a second DNA sequence bearing no complete lipoprotein structural gene and linked in reading phase with said first 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 the DNA sequence coding for the amino acid sequence of said at least one polypeptide.
Claim 4
Dependent Claim
153 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid comprising a first DNA sequence coding for at least one functional fragment derived from an outer membrane lipoprotein gene of Escherichia coli and selected from the group consisting of (a) the promoter, (b) the 5'-untranslated region, (c) the 3'-untranslated region and (d) the transcription termination signal of said lipoprotein gene, said first DNA sequence including at least the promoter of said lipoprotein gene, said plasmid further comprising a second DNA sequence bearing no complete lipoprotein structural gene and linked in reading phase with said first 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 the DNA sequence coding for the amino acid sequence of said at least one polypeptide.
Claim 5
Dependent Claim
123 words
A transformant derived from Escherichia coli comprising a plasmid comprised of a first DNA sequence coding for the amino acid sequence of a polypeptide foreign to said transformant, said first DNA sequence linked in reading phase with a second DNA sequence coding for at least one functional fragment derived from an outer membrane lipoprotein gene of Escherichia coli and selected from the group consisting of (a) the promoter, (b) the 5'-untranslated region, (c) the 3'-untranslated region and (d) the transcription termination signal of said lipoprotein gene, said second DNA sequence including at least the promoter of said lipoprotein gene, said transformant being capable of producing the polypeptide upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.
Claim 6
Dependent Claim
123 words
A transformant derived from Escherichia coli comprising a plasmid comprised of a first DNA sequence coding for the amino acid sequence of a polypeptide foreign to said transformant, said first DNA sequence linked in reading phase with a second DNA sequence coding for at least one functional fragment derived from an outer membrane lipoprotein gene of Escherichia coli and selected from the group consisting of (a) the promoter, (b) the 5'-untranslated region, (c) the 3'-untranslated region and (d) the transcription termination signal of said lipoprotein gene, said second DNA sequence including at least the promoter of said lipoprotein gene, said transformant being capable of producing the polypeptide upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.
Claim 7
Dependent Claim
160 words
A transformant derived from Escherichia coli comprising a plasmid comprised of a first DNA sequence coding for at least one functional fragment derived from an outer membrane lipoprotein gene of Escherichia coli and selected from the group consisting of (a) the promoter, (b) the 5'-untranslated region, (c) the 3'-untranslated region and (d) the transcription termination signal of said lipoprotein gene, said first DNA sequence including at least the promoter of said lipoprotein gene, said plasmid further comprising a second DNA sequence bearing no complete lipoprotein structural gene and linked in reading phase with said first 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 the DNA sequence coding for the amino acid sequence of a polypeptide foreign to said transformant, said transformant being capable of producing the polypeptide upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.
Claim 8
Dependent Claim
160 words
A transformant derived from Escherichia coli comprising a plasmid comprised of a first DNA sequence coding for at least one functional fragment derived from an outer membrane lipoprotein gene of Escherichia coli and selected from the group consisting of (a) the promoter, (b) the 5'-untranslated region, (c) the 3'-untranslated region and (d) the transcription termination signal of said lipoprotein gene, said first DNA sequence including at least the promoter of said lipoprotein gene, said plasmid further comprising a second DNA sequence bearing no complete lipoprotein structural gene and linked in reading phase with said first 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 the DNA sequence coding for the amino acid sequence of a polypeptide foreign to said transformant, said transformant being capable of producing the polypeptide upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.
Claim 9
Dependent Claim
112 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid comprising a first DNA sequence coding for the promoter and the 5'-untranslated region of an outer membrane lipoprotein gene of Escherichia coli, linked in reading phase with (a) a second DNA sequence located immediately downstream of said first DNA sequence and coding for the amino acid sequence of said at least one polypeptide, and (b) a third DNA sequence located immediately downstream of said second DNA sequence and coding for at least the 3'-untranslated region and the transcription termination signal of said lipoprotein gene.
Claim 10
Dependent Claim
112 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid comprising a first DNA sequence coding for the promoter and the 5'-untranslated region of an outer membrane lipoprotein gene of Escherichia coli, linked in reading phase with (a) a second DNA sequence located immediately downstream of said first DNA sequence and coding for the amino acid sequence of said at least one polypeptide, and (b) a third DNA sequence located immediately downstream of said second DNA sequence and coding for at least the 3'-untranslated region and the transcription termination signal of said lipoprotein gene.
Claim 11
Dependent Claim
136 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid comprising a first DNA sequence including DNA coding for the promoter and 5'-untranslated region and 3'-untranslated region and transcription termination signal of an outer membrane lipoprotein gene of Escherichia coli, linked in reading phase with a second DNA sequence bearing no complete lipoprotein structure gene and located downstream of said promoter and downstream of said 5'-untranslated region and upstream of said 3'-untranslated region and upstream of said transcription termination signal and coding for a translation initiation codon adjacent the recognition sequence of at least one restriction endonuclease for incorporation in said plasmid of the DNA sequence coding for the amino acid sequence of said at least one polypeptide.
Claim 12
Dependent Claim
136 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid comprising a first DNA sequence including DNA coding for the promoter and 5'-untranslated region and 3'-untranslated region and transcription termination signal of an outer membrane lipoprotein gene of Escherichia coli, linked in reading phase with a second DNA sequence bearing no complete lipoprotein structure gene and located downstream of said promoter and downstream of said 5'-untranslated region and upstream of said 3'-untranslated region and upstream of said transcription termination signal and coding for a translation initiation codon adjacent the recognition sequence of at least one restriction endonuclease for incorporation in said plasmid of the DNA sequence coding for the amino acid sequence of said at least one polypeptide.
Claim 13
Dependent Claim
36 words
A plasmid in accordance with claim 1 wherein said second DNA sequence further codes for a peptide extension capable of directing translocation of said at least one polypeptide across the cytoplasmic membrane of the bacterial host.
Claim 14
Dependent Claim
22 words
A plasmid in accordance with claim 7 wherein said first DNA sequence lies within the DNA sequence coding for the peptide extension.
Claim 15
Dependent Claim
26 words
A plasmid in accordance with claim 7 wherein said first DNA sequence lies at the 3' terminus of the DNA sequence coding for the peptide extension.
Claim 16
Dependent Claim
32 words
A plasmid in accordance with claim 7 wherein said second DNA sequence further codes for at most of the entire amino acid sequence of an outer membrane protein of a gram-negative bacterium.
Claim 17
Dependent Claim
35 words
A plasmid in accordance with claim 10 wherein said first DNA sequence 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 18
Dependent Claim
17 words
A plasmid in accordance with claim 1 wherein said at least one polypeptide comprises a mammalian hormone.
Claim 19
Dependent Claim
14 words
A plasmid in accordance with claim 12 wherein said mammalian hormone is human proinsulin.
Claim 20
Dependent Claim
11 words
A plasmid in accordance with claim 13 which is pInsulin A2-1.
Claim 21
Dependent Claim
36 words
A plasmid in accordance with claim 2 wherein said first DNA sequence further codes for a peptide extension capable of directing translocation of said at least one polypeptide across the cytoplasmic membrane of the bacterial host.
Claim 22
Dependent Claim
23 words
A plasmid in accordance with claim 15 wherein said second DNA sequence lies within said first DNA sequence coding for the peptide extension.
Claim 23
Dependent Claim
27 words
A plasmid in accordance with claim 15 wherein said second DNA sequence lies at the 3' terminus of said first DNA sequence coding for the peptide extension.
Claim 24
Dependent Claim
31 words
A plasmid in accordance with claim 15 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 25
Dependent Claim
36 words
A plasmid in accordance with claim 18 wherein said second DNA sequence lies within said first DNA sequence coding for at most the entire amino acid sequence of an outer membrane protein of a gram-negative bacterium.
Claim 26
Dependent Claim
28 words
A plasmid in accordance with claim 2 wherein said second DNA sequence codes for the recognition sequences for the Eco RI, Hind III and Bam HI restriction endonucleases.
Claim 27
Dependent Claim
17 words
A plasmid in accordance with claim 20 wherein said at least one polypeptide comprises a mammalian hormone.
Claim 28
Dependent Claim
14 words
A plasmid in accordance with claim 21 wherein said mammalian hormone comprises human proinsulin.
Claim 29
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN037.
Claim 30
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN039.
Claim 31
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN040.
Claim 32
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN041.
Claim 33
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN047.
Claim 34
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN048.
Claim 35
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN042.
Claim 36
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN043.
Claim 37
Dependent Claim
8 words
The plasmid of claim 22 which is pKEN044.
Claim 38
Dependent Claim
11 words
The transformant of claim 3 wherein said polypeptide is human proinsulin.
Claim 39
Dependent Claim
11 words
The transformant of claim 31 wherein said plasmid is pInsulin A2-1.
Claim 40
Dependent Claim
11 words
The transformant of claim 4 wherein said polypeptide is human proinsulin.
Claim 41
Dependent Claim
37 words
A plasmid in accordance with claim 5 wherein said third DNA sequence further includes a terminal portion of the structural sequence of said lipoprotein gene, and wherein said terminal portion is located upstream of said 3'-untranslated region.
Claim 42
Dependent Claim
37 words
A plasmid in accordance with claim 35 wherein said terminal portion is chosen so that the mRNA transcript of said third DNA sequence includes nucleotide sequences which permit the formation of at least five stable secondary structures.
Claim 43
Dependent Claim
24 words
A plasmid in accordance with claim 36 wherein said terminal portion codes for the segment commencing with position +168 of the lipoprotein structural gene.
Claim 44
Dependent Claim
42 words
A plasmid in accordance with claim 37 wherein said first DNA sequence further includes DNA coding for the peptide extension of said lipoprotein gene capable of directing translocation of said at least one polypeptide across the cytoplasmic membrane of the bacterial host.
Claim 45
Dependent Claim
24 words
A plasmid in accordance with claim 38 wherein said second DNA sequence is located within said first DNA sequence coding for the peptide extension.
Claim 46
Dependent Claim
28 words
A plasmid in accordance with claim 38 wherein said second DNA sequence is located at the 3' terminus of said first DNA sequence coding for the peptide extension.
Claim 47
Dependent Claim
45 words
A plasmid in accordance with claim 38 wherein said first DNA sequence further includes DNA coding for at least the first eight amino acid residues of the lipoprotein of Escherichia coli, linked with and located immediately downstream of the DNA coding for said peptide extension.
Claim 48
Dependent Claim
37 words
A plasmid in accordance with claim 41 wherein said second DNA sequence is located at the 3' terminus of the DNA coding for at least the first eight amino acid residues of the liproprotein of Escherichia coli.
Claim 49
Dependent Claim
17 words
A plasmid in accordance with claim 37 wherein said at least one polypeptide comprises a mammalian hormone.
Claim 50
Dependent Claim
26 words
A plasmid in accordance with claim 6 wherein said first DNA sequence further includes DNA coding for the peptide extension of the prolipoprotein of Escherichia coli.
Claim 51
Dependent Claim
24 words
A plasmid in accordance with claim 44 wherein said second DNA sequence is located within said first DNA sequence coding for said peptide extension.
Claim 52
Dependent Claim
28 words
A plasmid in accordance with claim 44 wherein said second DNA sequence is located at the 3' terminus of said first DNA sequence coding for said peptide extension.
Claim 53
Dependent Claim
40 words
A plasmid in accordance with claim 44 wherein said first DNA sequence further includes DNA coding for the first eight amino acid residues of said lipoprotein, linked with and located immediately downstream of the DNA coding for said peptide extension.
Claim 54
Dependent Claim
30 words
A plasmid in accordance with claims 45, 46 or 47 wherein said second DNA sequence comprises DNA sequences recognized by the Eco RI, Hind III and Bam HI restriction endonucleases.
Claim 55
Dependent Claim
44 words
A plasmid in accordance with claim 48 wherein said first DNA sequence further includes a terminal portion of the structural sequence of said lipoprotein gene, and wherein said terminal portion is located downstream of said second DNA sequence and upstream of said 3'-untranslated region.
Claim 56
Dependent Claim
36 words
A plasmid in accordance with claim 49 wherein said terminal portion is chosen so that the mRNA transcript of said first DNA sequence includes nucleotide sequences which permit formation of at least five stable secondary structures.
Claim 57
Dependent Claim
24 words
A plasmid in accordance with claim 50 wherein said terminal portion codes for the segment commencing with position +168 of the lipoprotein structural gene.
Claim 58
Dependent Claim
35 words
A plasmid in accordance with claim 47 wherein said second DNA sequence is located at the 3' terminus of the DNA coding for the first eight amino acid residues of the lipoprotein of Escherichia coli.
Claim 59
Dependent Claim
44 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid selected from the group consisting of pKEN037, pKEN039, pKEN040, pKEN041, pKEN047, pKEN048, pKEN042, pKEN043 and pKEN044.
Claim 60
Dependent Claim
44 words
A recombinant plasmid suited for use as a cloning vehicle for expression in a transformed bacterial host of at least one polypeptide foreign to Escherichia coli, said plasmid selected from the group consisting of pKEN037, pKEN039, pKEN040, pKEN041, pKEN047, pKEN048, pKEN042, pKEN043 and pKEN044.
Claim 61
Dependent Claim
51 words
A transformant derived from Escherichia coli for production of a polypeptide foreign to said transformant, said transformant chosen from the group consisting of E. coli JA221/pKEN037, E. coli JA221/pKEN039, E. coli JA221/pKEN040, E. coli JA221/pKEN041, E. coli JA221/pKEN047, E. coli JA221/pKEN048, E. coli JA221/pKEN042, E. coli JA221/pKEN043 and E. coli JA221/pKEN044.
Claim 62
Dependent Claim
51 words
A transformant derived from Escherichia coli for production of a polypeptide foreign to said transformant, said transformant chosen from the group consisting of E. coli JA221/pKEN037, E. coli JA221/pKEN039, E. coli JA221/pKEN040, E. coli JA221/pKEN041, E. coli JA221/pKEN047, E. coli JA221/pKEN048, E. coli JA221/pKEN042, E. coli JA221/pKEN043 and E. coli JA221/pKEN044.
Claim 63
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN037.
Claim 64
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN039.
Claim 65
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN040.
Claim 66
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN041.
Claim 67
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN047.
Claim 68
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN048.
Claim 69
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN042.
Claim 70
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN043.
Claim 71
Dependent Claim
10 words
The transformant of claim 54 which is E. coli JA221/pKEN044.
References Cited (Backward Citations)
US4237224A
Cited
EP0001929A2
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-11-24 | 1990-05-26 | 1990-11-25 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-11-24 | 1994-05-26 | 1994-11-25 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-11-24 | 1998-05-26 | 1998-11-25 | $7700.00 | Window Closed / Lapsed |