Patent US4801537A successfully refreshed from Google Patents.
US4801537A
Expired / Lapsed
US
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Vector for expression of polypeptides in bacilli
Assignee: Genex Corp
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Filing: 1985-03-29
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Granted: 1989-01-31
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Expires: 2005-03-29
Abstract
Abstract A replicable plasmidic expression vector, capable of high levels of expression and secretion of polypeptides in a bacillus is disclosed. The vector contains a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of proteases in a bacillus operably liked to a DNA sequence encoding the amino acid sequence of a polypeptide. The expression vector is particularly useful in the production of B. amyloliquefaciens proteases or other heterologous proteins in B. subtilis.
Inventors
- 👤 Vasantha Nagarajan
- 👤 Craig S. Rhodes
- 👤 Carl D. B. Banner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 20:50
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/195; C07K14/305; C07K14/31; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/625; C12N15/63; C12N15/70; C12N15/74; C12N15/75; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/52; C12N9/54; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/036; C07K2319/61; C07K2319/70; C07K2319/705; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/832; Y10S435/839; Y10S930/00; Y10S930/01; Y10S930/30
Claims Analysis
Total Claims: 64
Claim 1
Independent Claim
79 words
A process for producing a microorganism for the expression of a protease enzyme which comprises transforming calls of a first Bacillus species with a replicable plasmidic expression vector comprising: (a) a replicon; (b) a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of a protease in cells of a second Bacillus species; and (c) a DNA sequence encoding the amino acid sequence of the desired protease, operably linked to said promoter and regulatory regions.
Claim 2
Dependent Claim
79 words
A process for producing a microorganism for the expression of a protease enzyme which comprises transforming calls of a first Bacillus species with a replicable plasmidic expression vector comprising: (a) a replicon; (b) a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of a protease in cells of a second Bacillus species; and (c) a DNA sequence encoding the amino acid sequence of the desired protease, operably linked to said promoter and regulatory regions.
Claim 3
Dependent Claim
34 words
A process as claimed in claim 1, wherein the Bacillus which is transformed is a B. subtilis and the DNA sequences encoding the promoter and regulatory regions and the protease are B. amyloliquefaciens sequences.
Claim 4
Dependent Claim
24 words
A process as claimed in claim 2, wherein the B. amyloliquefaciens protease is selected from the group consisting of neutral protease and alkaline protease.
Claim 5
Dependent Claim
16 words
A process as claimed in claim 2, wherein said B. amyloliquefaciens protease is a neutral protease.
Claim 6
Dependent Claim
16 words
A process as claimed in claim 2, wherein said B. amyloliqufaciens protease is an alkaline protease.
Claim 7
Dependent Claim
45 words
A process as claimed in claim 2, 3, 4 or 5 wherein said B. subtilis contains a homologous plasmid having sufficient DNA sequence homology to sequences in said replicable plasmidic expression vector, whereby homologous recombination is capable between said homologous plasmid and said expression vector.
Claim 8
Dependent Claim
83 words
A replicable plasmidic expression vector capable of directing high level expression and secretion of a polypeptide in cells of a transformed Bacillus species which comprises: (a) a replicon; (b) a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of a Bacillus protease and wherein said DNA sequence is heterologous to and operable in said transformed Bacillus species; and (c) a DNA sequence encoding the amino acid sequence of a polypeptide, operably linked to said promoter and regulatory regions.
Claim 9
Dependent Claim
83 words
A replicable plasmidic expression vector capable of directing high level expression and secretion of a polypeptide in cells of a transformed Bacillus species which comprises: (a) a replicon; (b) a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of a Bacillus protease and wherein said DNA sequence is heterologous to and operable in said transformed Bacillus species; and (c) a DNA sequence encoding the amino acid sequence of a polypeptide, operably linked to said promoter and regulatory regions.
Claim 10
Dependent Claim
34 words
A replicable plasmidic expression vector as claimed in claim 7, wherein the DNA sequence comprising the promoter and regulatory regions is a sequence which controls the expression and secretion of a B. amyloliquefaciens protease.
Claim 11
Dependent Claim
18 words
A replicable plasmidic expression vector as claimed in claim 8, wherein said polypeptide is a B. amyloliquefaciens protease.
Claim 12
Dependent Claim
19 words
A replicable plasmidic expression vector as claimed in claim 8, wherein said polypeptide is a B. amyloliquefaciens neutral protease.
Claim 13
Dependent Claim
19 words
A replicable plasmidic expression vector as claimed in claim 8, wherein said polypeptide is a B. amyloliquefaciens alkaline protease.
Claim 14
Dependent Claim
3 words
The plasmid pGX2109.
Claim 15
Dependent Claim
3 words
The plasmid pGX2109.
Claim 16
Dependent Claim
3 words
The plasmid pGX2110.
Claim 17
Dependent Claim
3 words
The plasmid pGX2110.
Claim 18
Dependent Claim
3 words
The plasmid pGX2111.
Claim 19
Dependent Claim
3 words
The plasmid pGX2111.
Claim 20
Dependent Claim
15 words
A microorganism which has been transformed with the replicable plasmidic expression vector of claim 7.
Claim 21
Dependent Claim
19 words
A microorganism which has been transformed with the replicable plasmidic expression vector of claims 8, 9, 10 or 11.
Claim 22
Dependent Claim
14 words
A microorganism of a Bacillus species which has been transformed by the plasmid pGX2109.
Claim 23
Dependent Claim
14 words
A microorganism of a Bacillus species which has been transformed by the plasmid pGX2109.
Claim 24
Dependent Claim
14 words
A microorganism of a Bacillus species which has been transformed by the plasmid pGX2110.
Claim 25
Dependent Claim
14 words
A microorganism of a Bacillus species which has been transformed by the plasmid pGX2110.
Claim 26
Dependent Claim
14 words
A microorganism of a Bacillus species which has been transformed by the plasmid pGX2111.
Claim 27
Dependent Claim
14 words
A microorganism of a Bacillus species which has been transformed by the plasmid pGX2111.
Claim 28
Dependent Claim
17 words
A microorganism as claimed in claims 15, 17, 18 or 19 wherein said microorganism is B. subtilis.
Claim 29
Dependent Claim
14 words
A microorganism as claimed in claim 16 wherein said microorganism is a B. subtilis.
Claim 30
Dependent Claim
109 words
A mehod of producing a polypeptide which comprises: (a) transforming cells of a first Bacillus species with a replicable plasmidic expression vector comprising: (i) a replicon; p2 (ii) a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of a protease in cells of a second Bacillus species; and (iii) a DNA sequence encoding the amino acid sequence of a polypeptide heterolgous to and operably linked to said promoter and regulatory regions; (b) growing the transformed cells of a first Bacillus species in a culture medium and subjecting said transformed cells to conditions under which the polypeptide is expressed and secreted into the medium; and
Claim 31
Dependent Claim
109 words
A mehod of producing a polypeptide which comprises: (a) transforming cells of a first Bacillus species with a replicable plasmidic expression vector comprising: (i) a replicon; p2 (ii) a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of a protease in cells of a second Bacillus species; and (iii) a DNA sequence encoding the amino acid sequence of a polypeptide heterolgous to and operably linked to said promoter and regulatory regions; (b) growing the transformed cells of a first Bacillus species in a culture medium and subjecting said transformed cells to conditions under which the polypeptide is expressed and secreted into the medium; and
Claim 32
Dependent Claim
7 words
(c) recovering the polypeptide from the medium.
Claim 33
Dependent Claim
7 words
(c) recovering the polypeptide from the medium.
Claim 34
Dependent Claim
21 words
A method as claimed in claim 22, wherein the promoter and regulatory regions control expression of a protease in B. amyloliquefaciens.
Claim 35
Dependent Claim
17 words
A method as claimed in claim 23 wherein the Bacillus which is transformed is a B. subtilis.
Claim 36
Dependent Claim
15 words
A method as claimed in claim 24 wherein said polypeptide is a B. amyloliquefaciens protease.
Claim 37
Dependent Claim
16 words
A method as claimed in claim 24 wherein said polypeptide is a B. amyloliquefaciens neutral protease.
Claim 38
Dependent Claim
16 words
A method as claimed in claim 24 wherein said polypeptide is a B. amyloliquefaciens alkaline protease.
Claim 39
Dependent Claim
17 words
A method as claimed in claim 24 wherein said replicable plasmidic expression vector is the plasmid pGX2109.
Claim 40
Dependent Claim
17 words
A method as claimed in claim 24 wherein said replicable plasmidic expression vector is the plasmid pGX2110.
Claim 41
Dependent Claim
17 words
A method as claimed in claim 24 wherein said replicable plasmidic expression vector is the plasmid pGX2111.
Claim 42
Dependent Claim
65 words
A replicable plasmidic expression and secretion vector comprising the promotor and regulatory regions of a gene selected from the group consisting of the alkaline protease gene and neutral protease gene from B. amyloliquefaciens strain P and wherein said gene having fused, inframe, to the 3' end of the signal peptide coding sequence thereof, a structural gene encoding a polypeptide other than a B. amyloliquefaciens protease.
Claim 43
Dependent Claim
65 words
A replicable plasmidic expression and secretion vector comprising the promotor and regulatory regions of a gene selected from the group consisting of the alkaline protease gene and neutral protease gene from B. amyloliquefaciens strain P and wherein said gene having fused, inframe, to the 3' end of the signal peptide coding sequence thereof, a structural gene encoding a polypeptide other than a B. amyloliquefaciens protease.
Claim 44
Dependent Claim
46 words
A replicable plasmidic expression and secretion vector as claimed in claim 31, wherein the structural gene which is fused to the promotor and regulatory region contains at least a portion of the prorennin gene sequence and wherein said gene sequence encodes a polypeptide having prorennin activity.
Claim 45
Dependent Claim
28 words
A replicable plasmidic expression and secretion vector as claimed in claim 32, wherein the protein containing at least a portion of the prorennin gene sequence is delta 2-prorennin.
Claim 46
Dependent Claim
48 words
A replicable plasmidic expression and secretion vector as claimed in claim 31, wherein the structural gene which is fused to the promotor and regulatory region contains at least a portion of the protein A gene sequence and wherein said gene sequence encodes a polypeptide having protein A activity.
Claim 47
Dependent Claim
30 words
A replicable plasmidic expression and secretion vector as claimed in claim 34, wherein the protein containing at least a portion of the protein A gene sequence is delta 22-protein A.
Claim 48
Dependent Claim
29 words
A microorganism of the genus and species Bacillus subtilis which has been transformed with the replicable plasmidic expression and secretion vector of claims 31, 32, 33, 34 or 35.
Claim 49
Dependent Claim
73 words
An expression and secretion vector comprising a replicable plasmid containing the promoter and regulatory regions of a gene selected from the group consisting of the alkaline protease gene and neutral protease gene from B. amyloliquefaciens strain P and a sequence of deoxyribonucleotides, adjacent the 3' end of the signal peptide coding sequence, which comprises an endonuclease restriction site not naturally present in the alkaline or neutral protease genes from B. amyloliquefaciens strain P.
Claim 50
Dependent Claim
73 words
An expression and secretion vector comprising a replicable plasmid containing the promoter and regulatory regions of a gene selected from the group consisting of the alkaline protease gene and neutral protease gene from B. amyloliquefaciens strain P and a sequence of deoxyribonucleotides, adjacent the 3' end of the signal peptide coding sequence, which comprises an endonuclease restriction site not naturally present in the alkaline or neutral protease genes from B. amyloliquefaciens strain P.
Claim 51
Dependent Claim
22 words
A vector as claimed in claim 37, wherein the promoter and regulatory regions are from the alkaline protease gene from B. amyloliquefaciens.
Claim 52
Dependent Claim
26 words
A vector as claimed in claim 38, wherein the endonuclease restriction site adjacent the 3' end of the signal peptide coding sequence is a BamHI site.
Claim 53
Dependent Claim
2 words
Plasmid pGX2134.
Claim 54
Dependent Claim
2 words
Plasmid pGX2134.
Claim 55
Dependent Claim
22 words
A vector as claimed in claim 37, wherein the promoter and regulatory regions are from the neutral protease genes from B. amyloliquefaciens.
Claim 56
Dependent Claim
26 words
A vector as claimed in claim 41, wherein the endonuclease restriction site adjacent the 3' end of the signal peptide coding sequence is a BamHI site.
Claim 57
Dependent Claim
2 words
Plasmid pGX2138.
Claim 58
Dependent Claim
2 words
Plasmid pGX2138.
Claim 59
Independent Claim
101 words
A method of producing a polypeptide which comprises: (a) transforming cells of a first Bacillus species with a replicable plasmidic expression vector comprising: (i) a replicon; (ii) a DNA sequence comprising the promoter and regulatory regions which control, expression and secretion of neutral protease in cells of a first Bacillus species; and (iii) a DNA sequence encoding the amino acid sequence of a polypeptide heterologous to and operably linked to said promoter and regulatory regions; (b) growing the transformed cells of a first Bacillus species in culture medium; and (c) expressing the heterologous polypeptide during exponential growth of said transformed cells.
Claim 60
Independent Claim
101 words
A method of producing a polypeptide which comprises: (a) transforming cells of a first Bacillus species with a replicable plasmidic expression vector comprising: (i) a replicon; (ii) a DNA sequence comprising the promoter and regulatory regions which control, expression and secretion of neutral protease in cells of a first Bacillus species; and (iii) a DNA sequence encoding the amino acid sequence of a polypeptide heterologous to and operably linked to said promoter and regulatory regions; (b) growing the transformed cells of a first Bacillus species in culture medium; and (c) expressing the heterologous polypeptide during exponential growth of said transformed cells.
Claim 61
Dependent Claim
27 words
A method as claimed in claim 44, wherein the promoter and regulator regions are the promoter and regulatory regions of the neutral protease genes from B. amyloliquefaciens.
Claim 62
Independent Claim
96 words
A method of producing a polypeptide which comprises: (a) transforming a bacillus with a replicable plasmidic expression and secretion vector comprising: (i) a replicon; (ii) a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of alkaline protease cells of a first Bacillus species; and (iii) a DNA sequence encoding the amino acid sequence of a polypeptide heterologous to and operably linked to said promoter and regulatory regions; (b) growing said transformed cell in a culture medium; and (c) expressing the heterologous polypeptide after exponential growth of said transformed cells has ceased.
Claim 63
Independent Claim
96 words
A method of producing a polypeptide which comprises: (a) transforming a bacillus with a replicable plasmidic expression and secretion vector comprising: (i) a replicon; (ii) a DNA sequence comprising the promoter and regulatory regions which control expression and secretion of alkaline protease cells of a first Bacillus species; and (iii) a DNA sequence encoding the amino acid sequence of a polypeptide heterologous to and operably linked to said promoter and regulatory regions; (b) growing said transformed cell in a culture medium; and (c) expressing the heterologous polypeptide after exponential growth of said transformed cells has ceased.
Claim 64
Dependent Claim
27 words
A method as claimed in claim 46, wherein the promoter and regulatory regions are the promoter and regulatory regions of the alkaline protease genes from B. amyloliquefaciens.
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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) | 1992-01-31 | 1992-08-01 | 1993-01-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-01-31 | 1996-08-01 | 1997-01-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-01-31 | 2000-08-01 | 2001-01-31 | $7700.00 | Window Closed / Lapsed |