Cloning and expression of the gene for bacteriophage T7 RNA polymerase

• US 4,952,496 A
• Filed: 12/29/1986
• Issued: 08/28/1990
• Est. Priority Date: 03/30/1984
• Status: Expired due to Term

• Current Assignee: Brookhaven Science Associates, LLC (Government of the United States of America)
• Original Assignee: Associated Universities, Inc.
• Inventors: Moffatt, Barbara A., Dunn, John J., Studier, F. William, Rosenberg, Alan H., Davanloo, Parichehre
• Primary Examiner(s): Mays, Thomas
• Application Number:
  US07/002,408
• Time in Patent Office: 1,338 Days
• Field of Search: 435/68, 435/70, 435/91, 435/653, 435/235, 435/240.1, 435/176.3, 435/317.1, 435/320, 536/27, 935/6, 935/29, 935/32, 935/34, 935/38, 935/41, 935/50, 935/57, 935/60
• US Class Current: 435/91.41
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/73 Expression systems using phage (lambda) regulatory sequences
• CPC Class Codes: C12N 9/1247 DNA-directed RNA polymerase (2.7.7.6)

Abstract

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Claims

1. A process for obtaining clones of a DNA sequence encoding an enzymatically active RNA polymerase of any T7-like bacteriophage, comprising the steps of:

obtaining from the DNA of said T7-like bacteriophage a fragment that contains the complete coding sequence of a gene for said bacteriophage RNA polymerase;

eliminating from the fragment, or inactivating, any promoters that may lie to the 5'"'"' side of the 5'"'"' terminus of said coding sequence and which could direct the synthesis by any host cell RNA polymerase of active mRNA from the cloned gene;

eliminating from the fragment, or inactivating, any promoters that lie to the 5'"'"' side of the 5'"'"' terminus, to the 3'"'"' side of the 3'"'"' terminus and between said termini of said coding sequence and which are recognizable by said phage RNA polymerase, but retaining within the DNA fragment a sequence encoding an enzymatically active phage RNA polymerase; and

cloning said DNA fragment containing a sequence encoding enzymatically active phage RNA polymerase, but not containing functional promoters recognizable by said phage RNA polymerase, in a plasmid or other vector that contains no promoters recognizable by said phage RNA polymerase, and at a site in the vector where functional mRNA for said phage RNA polymerase would be only minimally transcribed by any host cell RNA polymerase.

2. The process of claim 1 applied to bacteriophage T7 to obtain clones of a DNA sequence encoding an enzymatically active T7 RNA polymerase.

9. The process of claim 1 wherein the T7-like phages are selected from the group consisting of Escherichia coli phages T3, φ

I, φ

II, W31, H, Y, A1122, cro, C21, C22 and C23;

Pseudomonas putida phage gh-1;

Salmonella typhimurium phage Sp6;

Serratia marcescens phage IV;

Citrobacter phage ViIII; and

Klebsiella phage number 11.

10. A process for producing enzymatically active RNA polymerase of a T7-like bacteriophage, comprising the steps of:

obtaining a DNA fragment containing a sequence encoding said enzymatically active bacteriophage RNA polymerase but not containing functional promoters recognizable by said bacteriophage RNA polymerase according to the process of claim 1;

placing said DNA fragment under control of a promoter that is effective for directing the synthesis of mRNA that can be translated to produce said enzymatically active bacteriophage RNA polymerase in a suitable host cell;

providing said appropriately controlled coding sequence to a host cell suitable for the production of said enzymatically active bacteriophage RNA polymerase; and

incubating under conditions that allow production, accumulation, and isolation of said enzymatically active bacteriophage RNA polymerase.

11. The process of claim 10 wherein the T7-like bacteriophage is selected from the group consisting of Escherichia coli phages T7, T3, φ

I, φ

II, W31, H, Y, A1122, cro, C21, C22, and C23;

Pseudomonas putida phage gh-1;

Salmonella typhimurium phage SP6;

Serratia marcescens phage IV;

Citrobacter phage ViIII; and

Klebsiella phage number 11.

12. The process of claim 10 wherein the T7-like phage is T7;

the cloned coding sequence is under control of the lacUV5 promoter in plasmid pAR1219;

the host cell is BL21 or other suitable host cell; and

conditions for production of enzymatically active T7 RNA polymerase include incubating said plasmid-containing host cell under conditions suitable for cell growth and production of proteins, and inducing the production of T7 RNA polymerase by adding a suitable concentration of IPTG or other inducer of the lacUV5 promoter.

13. A process for creating vectors to supply enzymatically active DNA polymerase of a T7-like bacteriophage to cells, comprising the steps of:

obtaining a DNA fragment containing a sequence encoding said enzymatically active bacteriophage RNA polymerase but not containing functional promoters recognizable by said bacteriophage RNA polymerase, according to the process of claim 1;

placing said DNA fragment in a selected vector and under control of a promoter that can direct the synthesis of mRNA that can be translated to produce said enzymatically active bacteriophage RNA polymerase in a desired host cell, the resulting vector being suitable for delivery to said desired host cell.

14. The process of claim 13 wherein said DNA fragment is the BamHI-ended DNA fragment from plasmid pAR1151, and said DNA fragment is inserted into the BamHI site of the lambda cloning vector D69.

15. Bacteriophage DE2, produced by the process of claim 14, and having a sequence encoding enzymatically active T7 RNA polymerase in the orientation to be expressed from the PI or PL promoters.

3. Plasmid pAR1151, ATCC 39561, containing a DNA sequence encoding enzymatically active T7 RNA polymerase, said plasmid comprising base pairs 3145 to 5841 of T7 DNA inserted into the BamHI site of pBR322 by use of synthetic linkers.

4. A BamHI-ended DNA fragment containing a sequence encoding enzymatically active T7 RNA polymerase which is obtainable from plasmid pAR1151, ATCC 39561.

5. Plasmid pAR1173, ATCC 39562, consisting of plasmid pAR1151 modified to contain a single BglII site to the 5'"'"' side of the 5'"'"' terminus of the DNA sequence encoding enzymatically active T7 RNA polymerase and within the BamHI-ended DNA fragment containing said coding sequence.

6. A BamHI-ended DNA fragment, containing a sequence encoding enzymatically active T7 RNA polymerase and a BglII site to the 5'"'"' side of the 5'"'"' terminus of said coding sequence and which is obtainable from plasmid pAR1173, ATCC 39562.

7. Plasmid pAR1219, ATCC 39563, consisting of a DNA fragment containing the lacI gene and the lacUV5 promoter of E.coli inserted into the BglII site of pAR1173 in the orientation such that transcription from the lacUV5 promoter produces functional mRNA encoding enzymatically active T7 RNA polymerase.

8. A BamHI-ended DNA fragment containing the lacI gene, the lacUV5 promoter, and a sequence encoding enzymatically active T7 RNA polymerase, and which is obtainable from plasmid pAR1219, ATCC 39563.

16. A process for creating vectors in which a DNA sequence encoding enzymatically active T7 RNA polymerase can be transcribed from a promoter recognizable by a selected host cell or viral RNA polymerase, comprising the steps of:

inserting said promoter into the BglII site of plasmid pAR1173, said promoter being oriented so as to produce mRNA encoding enzymatically active T7 RNA polymerase;

isolating the BamHI-ended DNA fragment containing said promoter and said coding sequence for T7 RNA polymerase; and

inserting said BamHI-ended DNA fragment into a selected vector suitable for establishment in or delivery to the desired host cell.

17. The process of claim 16 wherein said promoter is an inducible promoter.

18. The process of claim 17 wherein the BamHI-ended DNA fragment from plasmid pAR1219, is inserted into the BamHI site of the lambda cloning vector D69.

19. Bacteriophage DE3, produced by the process of claim 18, wherein the BamHI-ended DNA fragment from plasmid pAR1219 is in the orientation opposite to that in which functional mRNA encoding T7 RNA polymerase would be expressed from the PI or PL promoters.

20. A process for inserting the DNA sequence encoding enzymatically active T7 RNA polymerase into a bacterial chromosome, comprising the steps of:

making a lysogen of bacteriophage DE2 or DE3 in any bacterial cell capable of forming a lysogen of DE2 or DE3, under conditions that provide a source of active int protein.

21. The lysogens BL21(DE2), BL21(DE3), HMS174(DE2) and HMS174(DE3) formed by the process of claim 20.

22. Bacteriophage CE6, obtained from a genetic cross between DE2 and lambda cI857indlSam7, wherein said bacteriophage CE6 comprises a DNA sequence encoding enzymatically active T7 RNA polymerase and the cI857indlSam7 mutations.