EP0136090A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Improvements in or relating to expression plasmids
Assignee: EIDP Inc
•
Filing: 1984-08-24
•
Granted: —
•
Expires: 2004-08-24
Abstract
Abstract A promoter-operator Shine-Dalgarno Clal-containing fragment has been obtained comprising a -10 region and a Shine-Dalgarno sequence characteristic of the trp promoter-operator Shine-Dalgarno fragment and comprising in addition the following sequences: (i) a 6-base pair sequence in the -35 region of the fragment (5'-3') of and (ii) a sequence of about 17 base pairs between the -35 and the -10 regions of said fragment, the sequence of the first eight base pairs (5'-3') being Plasmids have been prepared comprising this promotor-operator fragment and plasmids have also been prepared comprising the trp promoter-operator, which are useful for the over-production in bacteria of particular proteins, such as β-lactamase, β-interferon and interleukin 2.
Inventors
- 👤 Rene Arentzen
- 👤 Stephen Robert Petteway, Jr.
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 06:24
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/70; C12N15/71; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2468; C12N9/2471; C12N9/78; C12N9/86; C12Y; C12Y302/00; C12Y302/01; C12Y302/01023; C12Y305/00; C12Y305/02; C12Y305/02006; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/70; C07K2319/74; C07K2319/75
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
77 words
A promoter-operator Shine-Dalgarno ClaI-containing fragment comprising about 600 base pairs and having a -10 region and a Shine-Dalgarno sequence characteristic of the trp promoter-operator Shine-Dalgarno fragment, and comprising the following sequences: (i) a 6-base pair sequence in the -35 region of the fragment (5'→ 3') of and (ii) a sequence of about 17 base pairs between the -35 and the -10 regions of said fragment. the sequence of the first eight base pairs (5' → 3') being
Claim 2
Dependent Claim
77 words
A promoter-operator Shine-Dalgarno ClaI-containing fragment comprising about 600 base pairs and having a -10 region and a Shine-Dalgarno sequence characteristic of the trp promoter-operator Shine-Dalgarno fragment, and comprising the following sequences: (i) a 6-base pair sequence in the -35 region of the fragment (5'→ 3') of and (ii) a sequence of about 17 base pairs between the -35 and the -10 regions of said fragment. the sequence of the first eight base pairs (5' → 3') being
Claim 3
Dependent Claim
9 words
A plasmid comprising a fragment according to Claim 1.
Claim 4
Dependent Claim
26 words
A plasmid according to Claim 2 comprising, additionally, one of: (i) a gene that codes for B-lactamase, or (ii) genes that code for IFN-B and B-lactamase.
Claim 5
Dependent Claim
68 words
A plasmid according to Claim 3, pKGP36, comprising a gene that codes for B-lactamase and a gene that codes for tetracycline resistance, the promoter-operator being oriented toward the tet R gene. the molecular weight of the plasmid being about 3. 0 x 10 6 daltons. the plasmid having the following partial restriction map: PstI-EcoRI, 750bp: EcoRI-ClaI. 23bp; ClaI-HpaI. 565bp; ClaI-ClaI. 600bp: HpaI-ClaI. 35bp: HpaI-HindIII. 39bp; HindIII-BamHI. 345 bp.
Claim 6
Dependent Claim
51 words
A plasmid according to Claim 2. pAP16. comprising a B-galactosidase gene lacking an initiating ATG codon. the promoter-operator being oriented toward said gene, the molecular weight of the plasmid being about 4.8 x 10 6 daltons, the plasmid having the following restriction map: PstI-HindIII. 1300bp; ClaI-ClaI. 600bp; HpaI-HindIII. 39bp: HpaI-BamHI. 45bp.
Claim 7
Dependent Claim
62 words
A plasmid according to claim 2, pAPFIF9, comprising a fused gene that codes for β-galactosidase and the amino terminus of IFN-β, the promoter- operator being oriented toward the IFN-β amino terminus coding sequence, the molecular weight of the plasmid being about 5.1x10 6 daltons, the plasmid having the following restriction map: PstI-HpaI, 1320bp; HpaI-HindIII, 39bp; HindIII-PstI, 207bp; PstI-HindIII, 270bp; and HindIII-BamHI, 6bp.
Claim 8
Dependent Claim
86 words
A plasmid according to Claim 2. selected from the group pKGP12 - 2. pKGP13-19. and pKGP9-25. comprising a fused gene that codes for B-galactosidase and the amino terminus of IFN-B, the gene coding for IFN-B having been edited to remove its signal sequence, the promoter-operator being oriented toward the edited IFN-B gene. the molecular weight of the plasmid being about 5.1 x 10 6 daltons. the plasmid having the following restriction map: PstI-HpaI. 1320 bp: FIpaI-ClaI, 27 bp: ClaI-PstI. 145 bp: PstI-HindIII. 270 bp: HindIII-BamHI. 6bp.
Claim 9
Dependent Claim
57 words
A plasmid according to Claim 2. selected from the group pKGP12-2R. pKGP13-19R. and pKGP9-25R. comprising a gene that codes for biologically active IFN-β, the promoter-operator being oriented toward the IFN-B gene, the molecular weight of the plasmid being about 3.4 x 10 6 daltons, the plasmid having the following restriction map: PstI-HpaI, 1330bp; HpaI-ClaI. 27bp: ClaI-PstI, 145bp.
Claim 10
Dependent Claim
60 words
A plasmid, pKGP43. comprising a gene that codes for B-lactamase and a gene that codes for tetracycline resistance, comprising also a tryptophan promoter-operator Shine-Dalgarno sequence oriented toward the ß-lactamase gene, the molecular weight of the plasmid being about 2.8 x 10 6 daltons, the plasmid having the following partial restriction map: PstI-EcoRI. 750bp; EcoRI-HpaI. 100bp: HpaI-HindIII, 300 bp: HindIII-BamHI. 345bp.
Claim 11
Dependent Claim
60 words
A plasmid, pKGP43. comprising a gene that codes for B-lactamase and a gene that codes for tetracycline resistance, comprising also a tryptophan promoter-operator Shine-Dalgarno sequence oriented toward the ß-lactamase gene, the molecular weight of the plasmid being about 2.8 x 10 6 daltons, the plasmid having the following partial restriction map: PstI-EcoRI. 750bp; EcoRI-HpaI. 100bp: HpaI-HindIII, 300 bp: HindIII-BamHI. 345bp.
Claim 12
Dependent Claim
51 words
A plasmid, pKGP13-19R·trp6, comprising a gene that codes for biologically active IFN-B, the plasmid having a tryptophan promoter-operator Shine-Dalgarno sequence oriented toward the IFN-B gene. the molecular weight of the plasmid being about 3.7 x 10 6 daltons, the plasmid having the following restriction map: PstI-HpaI, 1530bp: HpaI-ClaI, 27bp: ClaI-PstI, 145bp.
Claim 13
Dependent Claim
51 words
A plasmid, pKGP13-19R·trp6, comprising a gene that codes for biologically active IFN-B, the plasmid having a tryptophan promoter-operator Shine-Dalgarno sequence oriented toward the IFN-B gene. the molecular weight of the plasmid being about 3.7 x 10 6 daltons, the plasmid having the following restriction map: PstI-HpaI, 1530bp: HpaI-ClaI, 27bp: ClaI-PstI, 145bp.
Claim 14
Dependent Claim
54 words
A plasmid. pBREIL-2. comprising a gene that codes for B-lactamase and a gene that codes for IL-2. the molecular weight of the plasmid being about 1.7 x 10 6 daltons, the plasmid having the following partial restriction map: HindIII-IL-2 StuI site including IL-2 gene. 450 bp; IL-2 StuI site-HindIII excluding IL-2 gene. 2325 bp.
Claim 15
Dependent Claim
54 words
A plasmid. pBREIL-2. comprising a gene that codes for B-lactamase and a gene that codes for IL-2. the molecular weight of the plasmid being about 1.7 x 10 6 daltons, the plasmid having the following partial restriction map: HindIII-IL-2 StuI site including IL-2 gene. 450 bp; IL-2 StuI site-HindIII excluding IL-2 gene. 2325 bp.
Claim 16
Dependent Claim
68 words
A plasmid. pKGP36·trp, comprising a gene that codes for ß-lactamase and a gene that codes for tetracycline resistance, the trp promoter-operator being oriented toward the tet R gene. the molecular weight of the plasmid being about 3.0 X 1 0 6 daltons. the plasmid having the partial restriction map: HindIII-PstI. 3586 bp ; PstI-ClaI. 775 bp; ClaI - ClaI including Trp P.O.. 790 bp; Cla I-HindIII, 4 bp.
Claim 17
Dependent Claim
68 words
A plasmid. pKGP36·trp, comprising a gene that codes for ß-lactamase and a gene that codes for tetracycline resistance, the trp promoter-operator being oriented toward the tet R gene. the molecular weight of the plasmid being about 3.0 X 1 0 6 daltons. the plasmid having the partial restriction map: HindIII-PstI. 3586 bp ; PstI-ClaI. 775 bp; ClaI - ClaI including Trp P.O.. 790 bp; Cla I-HindIII, 4 bp.
Claim 18
Dependent Claim
55 words
13 . A plasmid. pTrpEIL-2. comprising a gene that codes for B-lactamase and a gene that codes for IL-2. the molecular weight of the plasmid being about 2. 1 x 10 6 daltons, the plasmid having the following partial restriction map: HindIII-Pst I including IL-2 gene. 2000 bp: Pst I-HindIII including Trp P.O.. 1569 bp.
Claim 19
Dependent Claim
55 words
13 . A plasmid. pTrpEIL-2. comprising a gene that codes for B-lactamase and a gene that codes for IL-2. the molecular weight of the plasmid being about 2. 1 x 10 6 daltons, the plasmid having the following partial restriction map: HindIII-Pst I including IL-2 gene. 2000 bp: Pst I-HindIII including Trp P.O.. 1569 bp.
Claim 20
Independent Claim
64 words
A method for making a plasmid selected from pKGP43 and pKGP36 as claimed in claims 4 and 9, comprising the steps of (i) restricting a plasmid of the pLD102 type using HpaII and Ball: (ii) isolating and purifying the trp-promoter-operator-containing HpaII/BalI fragment: (iii) modifying the HpaII/BalI fragment by limited digestion with TaqI: and (iv) inserting said modified fragment into the ClaI site of pBR322.
Claim 21
Dependent Claim
65 words
A method for making plasmid pAP16,as claimed in claim 5, comprising the steps of: (i) restricting a plasmid of the pLG400 type using PstI and HindIII to obtain a PstI/HindIII fragment; (ii) restricting a plasmid of the pKGP36 type with PstI and HindIII. and isolating and purifying the promoter/operator-containing fragment: and (iii) ligating the promoter-operator containing fragment of pKGP36 to the Pstl/HindIII fragment of pLG400.
Claim 22
Dependent Claim
83 words
A method for making plasmid pAPFIF9, as claimed in claim 6, comprising the steps of: (i) restricting the IFN-B cDNA fragment with HaeIII/HincII to obtain a HaeIII/HincII fragment, and isolating and purifying said fragment: (ii) ligating HindIII linkers to both ends of the HaeIII/HincII fragment of step (i) to obtain a HindIII/HindIII fragment, and isolating and purifying said fragment: (iii) restricting a plasmid of the pAP16 type using HindIII: and (iv) ligating the fragment of step (ii) with the fragment of step (iii).
Claim 23
Dependent Claim
142 words
A method for making a plasmid selected from pKGP12-2 and pKGP 13-19, as claimed in claim 7, comprising the steps of: (i) restricting a plasmid of the pAPl6 type using HindIII. and filling in the HindIII site with the Klenow fragment of E. coli DNA polymerase to obtain a blunt-ended fragment: (ii) restricting the fragment of step (i) using PstI to obtain a PstI fragment containing the amino terminal of the B-lactamase (AMP) gene and the pKGP36 tryptophan promoter-operator Shine-Dalgarno (P.O.-S.D.) unit, and purifying said fragment: (iii) restricting a plasmid of the pAPFIF9 type using PstI. and purifying the fragment which contains the carboxyl terminal of the B-lactamase gene and the carboxyl terminal of IFN-B cDNA: (iv) editing the IFN-B cDNA fragment giving a 140 base pair blunt ended ATG/PstI fragment: and (v) ligating the fragments of steps (ii), (iii) and (iv).
Claim 24
Dependent Claim
115 words
A method for making plasmid pKGP9-25, as claimed in claim 7, comprising the steps of: (i) restricting a plasmid of the pAP16 type using HindIII. and filling in the HindIII site with the Klenow fragment of E. coli DNA polymerase to obtain a blunt-ended fragment: (ii) digesting the fragment of step (i) with BamHI: (iii) restricting a plasmid of the pAPFIF9 type with HpaI to give three fragments: (iv) treating the fragments of step (iii) with Bal 31 exonuclease and BamHI to give fragments randomly degraded at the 5' end of the IFN-B gene and ending in an BamHI site: (v) ligating the IFN-B fragments of step (iv) into the pAP16 fragment of step (ii).
Claim 25
Dependent Claim
127 words
A method for making plasmids pKGP12-2R. pKGP13-19R, and pKG P9 - 25R , as claimed in claim 7, comprising the steps of: (i) restricting a plasmid of the pBR322 type using PstI and BamHI to obtain a PstI/BamHI fragment and purifying said fragment: (ii) restricting a plasmid of the (a) pKGP12-2. (b) pKGP13-19. or (c) pKGP9-25 type using PstI, and purifying the fragment which contains the amino terminal of B-lactamase the p KGP36 P.O.-S.D. unit and the edited amino terminal of the IFN-B gene: (iii) restricting a plasmid of the p3847 type using PstI and BgIII and purifying the fragment which contains the carboxyl terminal fragment of the IFN-B gene; and (iv) ligating the fragments of steps (i). (iii), and (a), (b) or (c) of step (ii).
Claim 26
Dependent Claim
94 words
A method for making plasmid pKGp13-19R·trp6, as claimed in claim 10, comprising the steps of: (i) restricting a plasmid of the pKGP43 type using HindIII and filling in the HindIII sites with the Klenow fragment of E. coli DNA polymerase to obtain a blunt-ended fragment; (ii) restricting the fragment of step (i) using HpaI and isolating the HpaI/ filled in HindIII fragment containing the -35 region of the trp promoter; (iii) restricting a plasmid of the pKGP13-19R type using HpaI; and (iv) ligating the fragment of step (ii) with the fragment of step (iii)
Claim 27
Dependent Claim
125 words
A method for making plasmid pBPEIL-2, as claimed in.claim 11, comprising the steps of: (i) restricting a plasmid of the plH40 type with PstI to produce a cDNA fragment containing the gene for IL-2: (ii) treating the fragment of step (i) to form a blunt 3° end beginning with the CCT codon: (iii) ligating to the blunt 3 1 end of the fragment of step (ii) an adapter molecule comprising ATG and GCT codons in serial arrangement and containing a HindIII/site; (iv) restricting the fragment of step (iii) with HindIII and StuI: (v) restricting a plasmid'of the pBR322 type with HindIII and PvuII, and isolating the large DNA fragment: (vi) ligating the IL-2 gene-containing fragment of step (iv) to the HindIII/PvuII fragment of step (v).
Claim 28
Dependent Claim
65 words
A method for making plasmid pKGP36-trp, as claimed in claim 12, comprising the steps of: (i) restricting a plasmid of the pKGP36 type with HpaI and PstI and purifying the fragment containing the tet R gene: (ii) restricting a plasmid of the pKGP13-19R*trp-6 type with HpaI and PstI, and purifying the fragment containing the trp P.O.; (iii) ligating the fragments of steps (i) and (ii).
Claim 29
Dependent Claim
64 words
A method for making plasmid pTrpEIL-2 as claimed in claim 13, comprising the steps of: (i) restricting a plasmid of the pBREIL-2 type with HindIII and PstI, and purifying the fragment containing the IL-2 gene: (ii) restricting a plasmid of the pKGP36·trp type with HindIII and PstI. and purifying the fragment containing the trp P.O.: (iii) ligating the fragments of steps (i) and (ii).
Claim 30
Dependent Claim
23 words
A mutated plasmid derived from a plasmid as claimed in any of claims 2 to 13 which is nevertheless a functional expression plasmid.
Claim 31
Dependent Claim
23 words
A method for modifying bacterial microorganisms which comprises inserting a plasmid as claimed in any of claims 2 to 13 and claim 24.
Claim 32
Dependent Claim
48 words
A method as claimed in claim 25 which comprises the steps of: (i) growing bacterial cells to a designated optical density: (ii) modifying the cell walls by treating with calcium chloride solution; (iii) mixing competent cells with ligated DNA mixture containing the plasmid: and (iv) incubating the mixture
Claim 33
Dependent Claim
18 words
A microorganism transformed by a plasmid as claimed in any of claims 2 to 13 and claim 24.
Claim 34
Dependent Claim
20 words
An E. coli bacterium transformed by a plasmid as claimed in any of claims 2 to 13 and claim 24.
Claim 35
Dependent Claim
57 words
A method for over-producing proteins from a transformed microorganism as claimed in claim 27, which comprises the steps of: (i) growing the transformed microorganism in an enriched medium to an optical density of at least 2 at 600 nm; (ii) diluting the grown cells 1:10 in minimal media; and (iii)treating the diluted cells with ß- indoleacrylic acid.
Claim 36
Dependent Claim
16 words
Molecule (A) comprising an initiation codon, the first codon of mature IL-2. and having the sequence:
Claim 37
Dependent Claim
16 words
Molecule (A) comprising an initiation codon, the first codon of mature IL-2. and having the sequence:
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.