Cloning and expression of the gene for bacteriophage T7 RNA polymerase

• US 5,869,320 A
• Filed: 01/15/1997
• Issued: 02/09/1999
• Est. Priority Date: 03/30/1984
• Status: Expired due to Fees

• Current Assignee: Brookhaven Science Associates, LLC (Government of the United States of America)
• Original Assignee: Brookhaven Science Associates, LLC (Government of the United States of America)
• Inventors: Studier, F. William, Moffatt, Barbara A., Rosenberg, Alan H., Davanloo, Parichehre, Dunn, John J.
• Primary Examiner(s): RAILEY, JOHNNY F
• Application Number:
  US08/784,201
• Time in Patent Office: 755 Days
• Field of Search: 435/320.1, 435/252.3, 435/69.1, 435/252.33, 530/350, 530/412, 536/23.72
• US Class Current: 435/252.33
• 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 R7 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. An isolated DNA sequence consisting of a cloned T7 RNA polymerase gene encoding an active T7 RNA polymerase.

2. A DNA expression vector encoding an active T7 RNA polymerase.

3. A cell transformed with the DNA expression vector of claim 2, wherein active T7 RNA polymerase is produced by the cell.

4. The cell of claim 3 which is a prokaryotic cell.

5. The cell of claim 4 which is E. coli.