US4783405A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Recombinant DNA expression vectors useful in bacillus and other host cells
Assignee: Eli Lilly and Co
•
Filing: 1984-09-26
•
Granted: 1988-11-08
•
Expires: 2004-09-26
Abstract
Abstract Recombinant DNA expression vectors for use in Bacillus and other host cells are disclosed. The vectors comprise the veg promoter sequence of Bacillus subtilis, a novel ribosome binding site-containing sequence and a sequence that codes for a functional polypeptide. The ribosome binding site-containing sequence is synthesized in accordance with conventional procedures while the veg promoter sequence can be obtained from E. coli K12 JA221/pMS480 (NRRL B-15258). Various sequences that codes for functional polypeptides and method for their expression in Bacillus are also disclosed.
Inventors
- 👤 Steven Kovacevic
- 👤 James R. Miller
- 👤 Hansen M. Hsiung
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 16:51
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/75; C12N15/76
Claims Analysis
Total Claims: 90
Claim 1
Independent Claim
106 words
A novel recombinant DNA expression vector which comprises (1) the ribosome binding site-containing DNA sequence ##STR9## wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, R is G or C, and R 1 is G or C, (2) the veg promoter of Bacillus subtilis, and (3) a gene that encoes a functional polypeptide, subject to the limitation that R and R 1 are not simultaneously the same deoxyribonucleotide and subject to the further limitation that said vector is selectable and that said promoter and said DNA sequence direct transcription and expression of said gene in a host cell transformed by said vector.
Claim 2
Dependent Claim
106 words
A novel recombinant DNA expression vector which comprises (1) the ribosome binding site-containing DNA sequence ##STR9## wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, R is G or C, and R 1 is G or C, (2) the veg promoter of Bacillus subtilis, and (3) a gene that encoes a functional polypeptide, subject to the limitation that R and R 1 are not simultaneously the same deoxyribonucleotide and subject to the further limitation that said vector is selectable and that said promoter and said DNA sequence direct transcription and expression of said gene in a host cell transformed by said vector.
Claim 3
Dependent Claim
10 words
The expression vector of claim 1 which is a plasmid.
Claim 4
Dependent Claim
58 words
The expression vector of claim 2 wherein the gene that encodes a functional polypeptide is selected from the group consisting of genes that encode human pre-proinsulin, human proinsulin, human insulin A-chain, human insulin B-chain, non-human insulin, human growth hormone, non-human growth hormone, bovine growth hormone, porcine growth hormone, human interferon, non-human inteferon, viral antigen, urokinase, hormones and enzymes.
Claim 5
Dependent Claim
13 words
The expression vector of claim 3 in which the gene encodes human pre-proinsulin.
Claim 6
Dependent Claim
13 words
The expression vector of claim 3 in which the gene encodes human proinsulin.
Claim 7
Dependent Claim
14 words
The expression vector of claim 3 in which the gene encodes human insulin A-chain.
Claim 8
Dependent Claim
14 words
The expression vector of claim 3 in which the gene encodes human insulin B-chain.
Claim 9
Dependent Claim
13 words
The expression vector of claim 3 in which the gene encodes non-human insulin.
Claim 10
Dependent Claim
14 words
The expression vector of claim 3 in which the gene encodes human growth hormone.
Claim 11
Dependent Claim
14 words
The expression vector of claim 3 in which the gene encodes non-human growth hormone.
Claim 12
Dependent Claim
14 words
The expression vector of claim 3 in which the gene encodes bovine growth hormone.
Claim 13
Dependent Claim
14 words
The expression vector of claim 3 in which the gene encodes porcine growth hormone.
Claim 14
Dependent Claim
13 words
The expression vector of claim 3 in which the gene encodes human interferon.
Claim 15
Dependent Claim
13 words
The expression vector of claim 3 in which the gene encodes viral antigen.
Claim 16
Dependent Claim
13 words
The expression vector of claim 3 in which the gene encodes a hormone.
Claim 17
Dependent Claim
13 words
The expression vector of claim 3 in which the gene encodes an enzyme.
Claim 18
Dependent Claim
36 words
The expression vector of claim 1 which is selected from the group consisting of plasmids pOW10, pOW303, pOW523, pOW524, POW525, pOW526, pO-W527, pOW528, pOW529, pOW530, pOW11, pOW531, pOW532, pOW533, pOW534, pOW310, pOW535, pOW536, pOW537 and pOW538.
Claim 19
Dependent Claim
9 words
The expression vector of claim 17 which is pOW10.
Claim 20
Dependent Claim
9 words
The expression vector of claim 17 which is pOW303.
Claim 21
Dependent Claim
9 words
The expression vector of claim 17 which is pOW523.
Claim 22
Dependent Claim
9 words
The expression vector of claim 17 which is pOW524.
Claim 23
Dependent Claim
9 words
The expression vector of claim 17 which is pOW525.
Claim 24
Dependent Claim
9 words
The expression vector of claim 17 which is pOW526.
Claim 25
Dependent Claim
9 words
The expression vector of claim 17 which is pOW527.
Claim 26
Dependent Claim
9 words
The expression vector of claim 17 which is pOW528.
Claim 27
Dependent Claim
9 words
The expression vector of claim 17 which is pOW529.
Claim 28
Dependent Claim
9 words
The expression vector of claim 17 which is pOW530.
Claim 29
Dependent Claim
49 words
The DNA sequence of claim 1 which is ##STR10## wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, R is G or C and R 1 is G or C, subject to the limitation that R and R 1 are not simultaneously the same deoxyribonucleotide.
Claim 30
Dependent Claim
13 words
A transformed host cell comprising a recombinant DNA expression vector of claim 1.
Claim 31
Dependent Claim
10 words
The transformed host cell of claim 29 which is Bacillus.
Claim 32
Dependent Claim
11 words
The transformed host cell of claim 30 which is Bacillus subtilis.
Claim 33
Dependent Claim
11 words
The transformed host cell of claim 29 which is E. coli.
Claim 34
Dependent Claim
59 words
The transformed host cell of claim 31 wherein the gene that encodes a functional polypeptide is selected from the group consisting of genes that encode human pre-proinsulin, human proinsulin, human insulin A-chain, human insulin B-chain, non-human insulin, human growth hormone, non-human growth hormone, bovine growth hormone, porcine growth hormone, human interferon, non-human interferon, viral antigen, urokinase, hormones and enzymes.
Claim 35
Dependent Claim
14 words
The transformed host cell of claim 33 in which the gene encodes human pre-proinsulin.
Claim 36
Dependent Claim
14 words
The transformed host cell of claim 33 in which the gene encodes human proinsulin.
Claim 37
Dependent Claim
15 words
The transformed host cell of claim 33 in which the gene encodes human insulin A-chain.
Claim 38
Dependent Claim
15 words
The transformed host cell of claim 33 in which the gene encodes human insulin B-chain.
Claim 39
Dependent Claim
15 words
The transformed host cell of claim 33 in which the gene encodes human growth hormone.
Claim 40
Dependent Claim
15 words
The transformed host cell of claim 33 in which the gene encodes bovine growth hormone.
Claim 41
Dependent Claim
15 words
The transformed host cell of claim 33 in which the gene encodes porcine growth hormone.
Claim 42
Dependent Claim
13 words
A transformed host cell comprising a recombinant DNA expression vector of claim 17.
Claim 43
Dependent Claim
38 words
The transformed host cell of claim 30 wherein the recombinant DNA expression vector is selected from the group consisting of plasmids pOW523, pOW524, POW525, pOW526, pOW527, pOW528, pOW529, pOW530, pOW531, pOW532, pOW533, pOW534, pOW535, pOW536, pOW537 and pOW538.
Claim 44
Dependent Claim
11 words
The transformed host cell of claim 41 which is E. coli.
Claim 45
Dependent Claim
11 words
The transformed host cell of claim 42 which is Bacillus subtilis.
Claim 46
Dependent Claim
12 words
The transformed host cell of claim 44 which is Bacillus subtilis MI112/pOW523.
Claim 47
Dependent Claim
12 words
The transformed host cell of claim 44 which is Bacillus subtilis MI112/pOW524.
Claim 48
Dependent Claim
12 words
The transformed host cell of claim 44 which is Bacillus subtilis MI112/pOW525.
Claim 49
Dependent Claim
12 words
The transformed host cell of claim 44 which is Bacillus subtilis MI112/pOW526.
Claim 50
Dependent Claim
12 words
The transformed host cell of claim 44 which is Bacillus subtilis MI112/pOW527.
Claim 51
Dependent Claim
12 words
The transformed host cell of claim 44 which is Bacillus subtilis MI112/pOW528.
Claim 52
Dependent Claim
12 words
The transformed host cell of claim 44 which is Bacillus subtilis MI112/pOW529.
Claim 53
Dependent Claim
12 words
The transformed host cell of claim 44 which is Bacillus subtilis MI112/pOW530.
Claim 54
Independent Claim
141 words
A method for producing a functional polypeptide in Bacillus wherein said polypeptide is secreted into the culture medium, said method comprises transforming a Bacillus host cell with a recombinant DNA expression vector which comprises (1) the ribosome binding site-containing DNA sequence ##STR11## wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, R is G or C, and R 1 is G or C (2) the veg promoter of Bacillus subtilis, and (3) a gene that encodes a signal peptide-containing functional polypeptide, and culturing said transformed Bacillus host cell under growth conditions, subject to the limitation that R and R 1 are not simultaneously the same deoxyribonucleotide and subject to the further limitation that said vector is selectable and that said promoter and said DNA sequence direct transcription and expression of said gene in said transformed Bacillus cell.
Claim 55
Independent Claim
141 words
A method for producing a functional polypeptide in Bacillus wherein said polypeptide is secreted into the culture medium, said method comprises transforming a Bacillus host cell with a recombinant DNA expression vector which comprises (1) the ribosome binding site-containing DNA sequence ##STR11## wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, R is G or C, and R 1 is G or C (2) the veg promoter of Bacillus subtilis, and (3) a gene that encodes a signal peptide-containing functional polypeptide, and culturing said transformed Bacillus host cell under growth conditions, subject to the limitation that R and R 1 are not simultaneously the same deoxyribonucleotide and subject to the further limitation that said vector is selectable and that said promoter and said DNA sequence direct transcription and expression of said gene in said transformed Bacillus cell.
Claim 56
Dependent Claim
19 words
The method of claim 53 wherein the expression vector is a plasmid and the host cell is Bacillus subtilis.
Claim 57
Dependent Claim
48 words
The method of claim 54 wherein the gene that encodes a signal peptide-containing polypeptide is selected from the group of genes consisting of genes that encode human pre-proinsulin, pre-growth hormone, pre-bovine growth hormone, pre-human growth hormone, pre-porcine growth hormone, immune modulators, proteolytic degradative enzymes, and cellulolytic degradative enzymes.
Claim 58
Dependent Claim
12 words
The method of claim 55 in which the gene encodes human pre-proinsulin.
Claim 59
Dependent Claim
12 words
The method of claim 55 in which the gene encodes pre-growth hormone.
Claim 60
Dependent Claim
13 words
The method of claim 55 in which the gene encodes pre-bovine growth hormone.
Claim 61
Dependent Claim
13 words
The method of claim 55 in which the gene encodes pre-porcine growth hormone.
Claim 62
Dependent Claim
13 words
The method of claim 55 in which the gene encodes pre-human growth hormone.
Claim 63
Dependent Claim
28 words
The method of claim 53 wherein the expression vector is selected from the group of plasmids consisting of plasmids pOW525, pOW526, pOW527, pOW528, POW531, pOW532, pOW533 and pOW534.
Claim 64
Dependent Claim
12 words
The method of claim 61 wherein the host cell is Bacillus subtilis.
Claim 65
Dependent Claim
14 words
The method of claim 62 in which the host cell is Bacillus subtilis MI112/pOW525.
Claim 66
Dependent Claim
14 words
The method of claim 62 in which the host cell is Bacillus subtilis MI112/pOW526.
Claim 67
Dependent Claim
14 words
The method of claim 62 in which the host cell is Bacillus subtilis MI112/pOW527.
Claim 68
Dependent Claim
14 words
The method of claim 62 in which the host cell is Bacillus subtilis MI112/pOW528.
Claim 69
Dependent Claim
10 words
The expression vector of claim 17 which is plasmid pOW11.
Claim 70
Dependent Claim
10 words
The expression vector of claim 17 which is plasmid pOW310.
Claim 71
Dependent Claim
10 words
The expression vector of claim 17 which is plasmid pOW531.
Claim 72
Dependent Claim
10 words
The expression vector of claim 17 which is plasmid pOW533.
Claim 73
Dependent Claim
10 words
The expression vector of claim 17 which is plasmid pOW535.
Claim 74
Dependent Claim
10 words
The expression vector of claim 17 which is plasmid pOW537.
Claim 75
Dependent Claim
12 words
The transformed host cell of claim 42 which is Bacillus subtilis MI112/pOW531.
Claim 76
Dependent Claim
12 words
The transformed host cell of claim 42 which is Bacillus subtilis MI112/pOW533.
Claim 77
Dependent Claim
12 words
The transformed host cell of claim 42 which is Bacillus subtilis MI112/pOW535.
Claim 78
Dependent Claim
12 words
The transformed host cell of claim 43 which is Bacillus subtilis MI112/pOW537.
Claim 79
Dependent Claim
13 words
The method of claim 61 wherein the host cell is Bacillus subtilis MII12/pOW531.
Claim 80
Dependent Claim
13 words
The method of claim 61 wherein the host cell is Bacillus subtilis MI112/pOW533.
Claim 81
Dependent Claim
16 words
An expression vector selected from the group consisting of plasmids pOW323/Z, pOW323, pOW324, pOW441 and pOW445.
Claim 82
Dependent Claim
16 words
An expression vector selected from the group consisting of plasmids pOW323/Z, pOW323, pOW324, pOW441 and pOW445.
Claim 83
Dependent Claim
8 words
The vector of claim 79 which is pOW323.
Claim 84
Dependent Claim
8 words
The vector of claim 77 which is pOW441.
Claim 85
Dependent Claim
8 words
The vector of claim 79 which is pOW445.
Claim 86
Dependent Claim
11 words
A transformed host cell comprising an expression vector of claim 79.
Claim 87
Dependent Claim
34 words
The transformed host cell of claim 83 which is selected from the group consisting of E. coli and Bacillus subtilis and wherein the plasmid is selected from the group consisting of pOW441 and pOW445.
Claim 88
Dependent Claim
12 words
The transformed host cell of claim 84 which is Bacillus subtilis MI112/pOW441.
Claim 89
Dependent Claim
12 words
The transformed host cell of claim 84 which is Bacillus subtilis MI112/pOW445.
Claim 90
Dependent Claim
11 words
The method of claim 53 wherein the expression vector is pOW441.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-11-08 | 1992-05-09 | 1992-11-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-11-08 | 1996-05-09 | 1996-11-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-11-08 | 2000-05-09 | 2000-11-08 | $7700.00 | Window Closed / Lapsed |