EP0036776A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
A method of creating an expression plasmid
Assignee: Genentech Inc
•
Filing: 1981-03-23
•
Granted: —
•
Expires: 2001-03-23
Abstract
Abstract Novel plasmidic expression vehicles and methods of using them in the production of useful polypeptides by recombinant bacteria are described. The plasmids employ a tryptophan promoter-operator system from which the attenuator region ordinarily present has been deleted. Bacteria containing-the plasmids can accordingly be repressed by the addition of tryptophan against expression of desired polypeptides coded for by inserted genes while they are grown to levels suitable for industrial-scale production. Additive tryptophan may then be withdrawn, essentially derepressing the pathway and permitting efficient production of the desired product in high yield.
Inventors
- 👤 Dennis G. Kleid
- 👤 Daniel G. Yansura
- 👤 Herbert L. Heyneker
- 👤 Giuseppe F. Miozzari
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-27 23:19
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/61; C07K14/655; C12N15/11; C12N15/62; C12N15/63; C12N15/70; C12N15/71; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/70; C07K2319/74; C07K2319/75
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
132 words
A method of creating an expression plasmid for the expression of a heterologous gene which comprises the simultaneous ligation, in phase, of: (a) a first linear double-stranded DNA fragment containing a replicon and a gene which expresses a Selectable characteristic when placed under the direction of a bacterial promoter, said fragment lacking any such promoter; (b) a second linear double-stranded DNA fragment comprising said heterologous gene; and (c) a third double-stranded DNA fragment which comprises a bacterial promoter; the ligatable ends of said fragments being configured such that upon ligation to form a replicable plasmid both the gene for the selectable characteristic and the heterologous gene come under the direction of the promoter, thus permitting use of the selectable characteristic in selection of transformant bacteria colonies capable of expressing the heterologous gene.
Claim 2
Independent Claim
132 words
A method of creating an expression plasmid for the expression of a heterologous gene which comprises the simultaneous ligation, in phase, of: (a) a first linear double-stranded DNA fragment containing a replicon and a gene which expresses a Selectable characteristic when placed under the direction of a bacterial promoter, said fragment lacking any such promoter; (b) a second linear double-stranded DNA fragment comprising said heterologous gene; and (c) a third double-stranded DNA fragment which comprises a bacterial promoter; the ligatable ends of said fragments being configured such that upon ligation to form a replicable plasmid both the gene for the selectable characteristic and the heterologous gene come under the direction of the promoter, thus permitting use of the selectable characteristic in selection of transformant bacteria colonies capable of expressing the heterologous gene.
Claim 3
Dependent Claim
12 words
The method of claim 1 wherein the selectable characteristic is antibiotic resistance.
Claim 4
Dependent Claim
21 words
The method of claim 2 wherein the selectable characteristic is tetracycline resistance and wherein the bacterial promoter is the trp promoter.
Claim 5
Dependent Claim
18 words
The method of claim 3 wherein ligation reconstitutes an operon for the expression of ampicillin resistance as well.
Claim 6
Independent Claim
114 words
A method of cleaving double stranded DNA at any given point which comprises: (a) converting the double stranded DNA to single-stranded DNA in a region surrounding said point; (b) hybridizing to the single-stranded region formed in step (a) a complementary primer length of single-stranded DNA, the 5' end of the primer lying . opposite the nucleotide adjoining the intended cleavage site; (c) restoring that portion of the second strand eliminated in step (a) which lies in the 3' direction from said primer by reaction with DNA polymerase in the presence of adenine, thymine, guanine and cytosine-containing deoxynucleotide triphosphates; and (d) digesting the remaining single-stranded length of DNA which protrudes beyond the intended cleavage point.
Claim 7
Independent Claim
114 words
A method of cleaving double stranded DNA at any given point which comprises: (a) converting the double stranded DNA to single-stranded DNA in a region surrounding said point; (b) hybridizing to the single-stranded region formed in step (a) a complementary primer length of single-stranded DNA, the 5' end of the primer lying . opposite the nucleotide adjoining the intended cleavage site; (c) restoring that portion of the second strand eliminated in step (a) which lies in the 3' direction from said primer by reaction with DNA polymerase in the presence of adenine, thymine, guanine and cytosine-containing deoxynucleotide triphosphates; and (d) digesting the remaining single-stranded length of DNA which protrudes beyond the intended cleavage point.
Claim 8
Dependent Claim
45 words
The method of claim 5 wherein steps (c) and (d) are performed simultaneously by reaction with DNA polymerase which polymerizes in the direction of 5' 4 3', is exonucleolytic in the direction of 3' 1 5', but non-exonucleolytic in the direction of 5' → 3'.
Claim 9
Dependent Claim
12 words
The method of claim 6 wherein the polymerase is Klenow Polymerase I.
Claim 10
Dependent Claim
107 words
A plasmidic expression vehicle for the production in E. coli bacteria of a heterologous polypeptide product, said vehicle having a sequence of double-stranded DNA comprising, in phase from a first 5' to a second 3' end of the coding strand thereof, the elements: (i) a bacterial trp promoter-operator system; (ii) nucleotides coding for a ribosome binding site for translation of element (iv); (iii) nucleotides coding for a translation start signal for translation of element (iv); and (iv) a structural gene encoding the amino acid sequence of a heterologous polypeptide; said sequence comprising neither any trp attenuation capability nor nucleotides coding for the trp E ribosome binding site.
Claim 11
Dependent Claim
107 words
A plasmidic expression vehicle for the production in E. coli bacteria of a heterologous polypeptide product, said vehicle having a sequence of double-stranded DNA comprising, in phase from a first 5' to a second 3' end of the coding strand thereof, the elements: (i) a bacterial trp promoter-operator system; (ii) nucleotides coding for a ribosome binding site for translation of element (iv); (iii) nucleotides coding for a translation start signal for translation of element (iv); and (iv) a structural gene encoding the amino acid sequence of a heterologous polypeptide; said sequence comprising neither any trp attenuation capability nor nucleotides coding for the trp E ribosome binding site.
Claim 12
Independent Claim
179 words
The method of producing a polypeptide product by the expression in bacteria of a structural gene coding therefor which comprises: (a) providing a bacterial inoculant transformed with a replicable plasmidic expression vehicle having a sequence of double-stranded DNA comprising, in phase from a first 5' to a second 3' end of the coding strand thereof, the elements: (i) a bacterial trp promoter-operator system; (ii) nucleotides coding for a ribosome binding site for translation of element (iv); (iii) nucleotides coding for a translation start signal for translation of element (iv); and (iv) a structural gene encoding the amino acid sequence of a heterologous polypeptide; said sequence comprising neither any trp attenuation capability nor nucleotides coding for the trp E ribosome binding site; (b) placing.the transformed inoculant in a fermentation vessel and growing the same to a predetermined level in suitable nutrient media containing additive tryptophan sufficient in quantity to repress said promoter-operator system; and (c) depriving said bacteria of said additive so as to derepress said system and occasion the expression of the product for which said structural gene codes.
Claim 13
Independent Claim
179 words
The method of producing a polypeptide product by the expression in bacteria of a structural gene coding therefor which comprises: (a) providing a bacterial inoculant transformed with a replicable plasmidic expression vehicle having a sequence of double-stranded DNA comprising, in phase from a first 5' to a second 3' end of the coding strand thereof, the elements: (i) a bacterial trp promoter-operator system; (ii) nucleotides coding for a ribosome binding site for translation of element (iv); (iii) nucleotides coding for a translation start signal for translation of element (iv); and (iv) a structural gene encoding the amino acid sequence of a heterologous polypeptide; said sequence comprising neither any trp attenuation capability nor nucleotides coding for the trp E ribosome binding site; (b) placing.the transformed inoculant in a fermentation vessel and growing the same to a predetermined level in suitable nutrient media containing additive tryptophan sufficient in quantity to repress said promoter-operator system; and (c) depriving said bacteria of said additive so as to derepress said system and occasion the expression of the product for which said structural gene codes.
Claim 14
Dependent Claim
21 words
The vehicle of claim 8 or method of claim 9 wherein the polypeptide expressed by said structural gene is entirely heterologous.
Claim 15
Dependent Claim
37 words
The vehicle of claim 8 or the method of claim 9 wherein the polypeptide expressed is a fusion protein comprising a heterologous polypeptide and at least a portion of the amino acid sequence of a homologous polypeptide.
Claim 16
Dependent Claim
31 words
The vehicle or method of claim 11. wherein said portion is a portion of the amino acid sequence of an enzyme involved in the biosynthetic pathway from chorismic acid to tryptophan.
Claim 17
Dependent Claim
26 words
The vehicle or method of claim 12 wherein the heterologous polypeptide is a bioactive polypeptide and the fused homologous polypeptide is a specifically cleavable bioinactivating polypeptide.
Claim 18
Dependent Claim
32 words
The vehicle or method of claim 11 wherein the homologous polypeptide is the trp E polypeptide and wherein said ribosome binding site is the ribosome binding site for the trp leader polypeptide.
Claim 19
Dependent Claim
16 words
The vehicle or method of claim 11 wherein the homologous polypeptide is the trp D polypeptide.
Claim 20
Dependent Claim
55 words
The vehicle or method of claim 14 wherein the fusion protein comprises an heterologous polypeptide and a homologous polypeptide which itself constitutes a fusion of about the first six amino acids of the trp leader polypeptide and the amino acid sequence encoded by at least about the distal third of the trp E polypeptide gene.
Claim 21
Dependent Claim
46 words
The vehicle or claim 8 or method of claim 9 wherein the heterologous polypeptide comprises a recoverable polypeptide selected from the group consisting of human growth hormone, human proinsulin, somatostatin, thymosin alpha 1, the A chain of human insulin and the B chain of human insulin.
Claim 22
Dependent Claim
32 words
The method of claim 8 wherein tryptophan deprivation is effected by cessation of addition of said additive and by dilution of the fermentation media in which said inoculant is first grown up.
Claim 23
Dependent Claim
12 words
The method of claim 18 wherein the host bacteria is E. coli.
Claim 24
Dependent Claim
10 words
The plasmids pBRHtrp, pSOM7A2, pHGH207, pHKYl, pSOM7Δ2Δ4, pThyα7Δ1Δ4, and pThα7Δ2.
Claim 25
Dependent Claim
10 words
The plasmids pBRHtrp, pSOM7A2, pHGH207, pHKYl, pSOM7Δ2Δ4, pThyα7Δ1Δ4, and pThα7Δ2.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.