EP0067540A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Microbial gene promoter/operators
Assignee: Genentech Inc
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Filing: 1982-05-18
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Granted: —
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Expires: 2002-05-18
Abstract
Abstract Hybrid promotenoperators are prepared and inserted into plasmids which are used to transform E. coli. They are arranged to control expression (by a gene in the plasmid) of a desired heterologous peptide e.g. human growth hormone, HGH. Each hybrid promoter/operator has (a) a promoter: operator fragment which is capable of being chemically induced, and (b) a fragment which includes the -35 consensus sequence and the 5' flanking region thereof of a promoter having a high signal strength. The fragments (a) and (b) are appropriately spaced to form an operable hybrid promoter which is both inducible and of high signal strength (properties not naturally found together). E.g. one hybrid operator promoter has a sequence from upstream of position -22 of the trp promoter (of high strength) coupled to a sequence downstream from position -19 of the lac promoter operator (inducible). This was used to control a gene for HGH in a plasmid pHGH807tacl. E. coli was transformed therewith and cultured. Production of HGH (rapid owing to the strong promoter fragment) was induced by addition of isopropyl-o-D-galactoside to the culture medium.
Inventors
- 👤 Herman Albert De Boer
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 01:50
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/70; C12N15/72; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/61
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
85 words
A hybrid promoter/operator capable of directing E. coli DNA-dependent RNA polymerase transcription of a recombinant DNA fragment positioned downstream therefrom comprising: a) a promoter/operator sequence capable of being derepressed by induction and exhibiting a first signal strength, and, operably positioned upstream therefrom, b) a fragment comprising the -35 consensus sequence and 5' flanking region of a second promoter having a greater signal strength; elements a) and b) being covalently linked between about said -35 consensus sequence and the Pribnow box of said promoter/ operator sequence.
Claim 2
Dependent Claim
85 words
A hybrid promoter/operator capable of directing E. coli DNA-dependent RNA polymerase transcription of a recombinant DNA fragment positioned downstream therefrom comprising: a) a promoter/operator sequence capable of being derepressed by induction and exhibiting a first signal strength, and, operably positioned upstream therefrom, b) a fragment comprising the -35 consensus sequence and 5' flanking region of a second promoter having a greater signal strength; elements a) and b) being covalently linked between about said -35 consensus sequence and the Pribnow box of said promoter/ operator sequence.
Claim 3
Dependent Claim
26 words
A hybrid promoter/operator according to claim 1 wherein said elements a) and b) are linked at a position between about nucleotide base pairs -10 to -35.
Claim 4
Dependent Claim
23 words
A hybrid p romoter/operator according to claim 1 or 2 wherein said elements a) and b) are linked via complementary restriction sites thereon.
Claim 5
Dependent Claim
15 words
A hybrid promoter/operator according to claim 3 wherein said complementary restriction sites are synthetically derived.
Claim 6
Dependent Claim
36 words
A hybrid promoter/operator according to claim 1, 2, 3 or 4 wherein said element a) is derived from a promoter/operator selected from the group consisting of lac, gal P2, ara BAD, ara C and gal Pl.
Claim 7
Dependent Claim
35 words
A hybrid promoter/operator according to claim 1, 2, 3, 4 or 5 wherein said element b) is derived from a promoter selected from the group consisting of trp, rrn, PR, PL, str, spc and rpoB.
Claim 8
Dependent Claim
54 words
A hybrid promoter/operator comprising the DNA sequence downstream from a position between about -10 to -35 of a promoter/operator whose operator is capable of being derepressed by induction, said DNA sequence being linked to a DNA sequence upstream from a position between about -10 to -35 of a promoter with a greater signal strength.
Claim 9
Dependent Claim
54 words
A hybrid promoter/operator comprising the DNA sequence downstream from a position between about -10 to -35 of a promoter/operator whose operator is capable of being derepressed by induction, said DNA sequence being linked to a DNA sequence upstream from a position between about -10 to -35 of a promoter with a greater signal strength.
Claim 10
Dependent Claim
27 words
A hybrid promoter/operator according to any of claims 1 to 7 wherein said promoter/operator whose operator is capable of being derepressed by induction is the lac promoter/operator.
Claim 11
Dependent Claim
32 words
A hybrid promoter/operator according to claim 8 comprising the DNA sequence downstream from position -19 of the lac promoter/operator linked to the DNA sequence upstream from position -22 of the trp promoter.
Claim 12
Dependent Claim
37 words
A hybrid promoter/operator according to claim 8 comprising the DNA sequence upstream from position -12 of the trp promoter linked to a DNA sequence patterned after the DNA sequence downstream from position -11 of the lac promoter.
Claim 13
Dependent Claim
40 words
A hybrid promoter/operator according to claim 8 comprising a DNA sequence downstream from position -19 of the lac promoter and a DNA sequence upstream from position -28 of the rrn promoter, linked via a synthetic DNA sequence of nucleotide bases.
Claim 14
Dependent Claim
53 words
A process for producing a hybrid promoter/opera:or according to any one of claims 1 to 11, which comprises covalently linking said promoter/operator sequence a) aid said fragment b) between about said -35 consensus sequence and the Pribnow box of said promoter/operator sequence so that fragment b) is operably positioned upstream from sequence a).
Claim 15
Dependent Claim
30 words
A replicable expression vehicle comprising a heterologous gene insert the expression of which is under the control of a hybrid promoter/operator according to any one of claims 1 to 11.
Claim 16
Dependent Claim
12 words
An E. coli strain transformed with a vehicle according to claim 13.
Claim 17
Dependent Claim
10 words
A plasmid selected from pHGH807-tacI, pBR322-Prrnlac, pHGH-Prac 5-16 and pHGH907-tacII.
Claim 18
Dependent Claim
10 words
A plasmid selected from pHGH807-tacI, pBR322-Prrnlac, pHGH-Prac 5-16 and pHGH907-tacII.
Claim 19
Dependent Claim
33 words
An E. coli culture capable of producing a heterologous polypeptide by expression of a gene coding therefor, said expression being directed by a hybrid promoter/operator according to any of claims 1 to 11.
Claim 20
Dependent Claim
10 words
A product produced by the culture according to claim 16.
Claim 21
Dependent Claim
46 words
Use of a hybrid promoter/operator of any one of claims 1 to 11 for producing a replicable expression vehicle according to claim 13, a transformed E. coli strain of claim 14, an E. coli culture according to claim 16, or a product according to claim 17.
Claim 22
Dependent Claim
34 words
Use of a replicable expression vehicle according to claim 13 for producing a transformed E. coli strain of claim 14, an E. coli culture of claim 16 or a product according to claim 17.
Claim 23
Dependent Claim
26 words
Use of a transformed E. coli strain of claim 14 to produce an E. coli culture of claim 16 or a product according to claim 17.
Claim 24
Dependent Claim
32 words
Use of a plasmid of claim 15 to produce a transformed E. coli strain of claim 14, an E. coli culture according to claim 16 or a product according to claim 17.
Claim 25
Dependent Claim
15 words
Use of an E. coli strain according to claim 16 to produce a heterologous polypeptide.
References Cited (Backward Citations)
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