Microbial hybrid promoters

• US 4,551,433 A
• Filed: 01/11/1982
• Issued: 11/05/1985
• Est. Priority Date: 05/18/1981
• Status: Expired due to Term

• Current Assignee: Genetec Incorporated (Noritsu Koki Co., Ltd.)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: DeBoer, Herman A.
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Martinell, James
• Application Number:
  US06/338,397
• Time in Patent Office: 1,394 Days
• Field of Search: 35/68, 35/70, 35/172, 35/253, 35/317, 35/849, 35/172.3, 35/71, 536/27
• US Class Current: 435/252.33
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: Y10S 435/849 Escherichia coli

Abstract

Novel microbial hybrid promoters and their use to direct the microbial expression of heterologous genes are described. Such promoters are selectively and functionally constructed by recombinant techniques, utilizing the discovery that certain DNA regions of given promoters are responsible for particularly advantageous functional properties.

Claims

1. 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 portion of a promoter/operator sequence which portion is capable of being derepressed by induction and which promoter exhibits a first strength, and,

(b) operably positioned upstream therefrom, a fragment comprising the -35 consensus sequence and 5'"'"' flanking region of a second promoter having a greater strength;

elements (a) and (b) being covalently linked between about said -35 consensus sequence and the Prinbnow box of said promoter/operator sequence.

2. 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.

3. A hybrid promoter/operator according to claim 1 wherein said elements (a) and (b) are linked via complementary restriction sites thereon.

4. A hybrid promoter/operator according to claim 3 wherein said complementary restriction sites are synthetically derived.

5. A hybrid promoter/operator according to claims 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 P1.

6. A hybrid promoter/operator according to claims 1, 2, 3, or 4 wherein said element (b) is derived from a promoter selected from the group consisting of trp, rrn, lambda PR, lambda PL, str, spc and rpoB.

7. 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 claim 1.

10. The hybrid promoter/operator of claims 1, 8, or 9 wherein the operator sequence is the operator sequence of the lac promoter/operator.

11. The hybrid promoter/operator of claim 10 which comprises the DNA sequence downstream from position -19 of the lac promoter/operator linked to, and downstream from, the DNA sequence upstream of position -22 of the trp promoter.

12. A hybrid promoter/operator according to claim 10 comprising a DNA sequence upstream from position -12 of the trp promoter linked to a DNA sequence which comprises the core of the lac operator.

13. The hybrid promoter/operator of claim 10 which comprises a DNA sequence downstream from position -19 of the lac promoter operator linked downstream from, and via a synthetic DNA sequence to, a DNA sequence upstream from position -28 of the rrn promoter.

14. A replicable expression vehicle comprising a heterologous gene insert, the expression of which is under the control of the hybrid promoter/operator of claims 1, 8, or 9.

15. An E.coli strain transformed with the vehicle according to claim 14.

8. A hybrid promoter/operator which comprises:

(a) an operator DNA sequence of a first promoter/operator which is capable of being derepressed by induction, and which comprises that sequence immediately downstream of a position between about -10 and -35 of said first promoter operator; and

(b) upstream from the sequence of (a), a DNA sequence which comprises that sequence of a second promoter which is immediately upstream of a position between about -10 and -35 of said second promoter.

9. A hybrid promoter/operator capable of directing E.coli DNA-dependent RNA polymerase catalyzed transcription which comprises:

(a) an upstream portion comprising the DNA sequence of a first promoter, which sequence is upstream of a position between about -10 and -35 of such first promoter; and

(b) a downstream portion comprising the DNA sequence of a second promoter/operator which sequence is downstream of a position between about -10 and -35 of said second promter/operator, and which sequence contains an operator which is capable of being derepressed by induction.