A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.

TLDR

T7 RNA polymerase’s high promoter specificity, together with rifampicin‑mediated host RNA polymerase inhibition, enables exclusive expression from T7 promoters. The study presents a coupled system to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase. The system uses a lambda PL‑promoter–driven plasmid to induce T7 RNA polymerase, which is then purified to homogeneity and protected from proteolysis by optimized lysis conditions. Cloning the T7 RNA polymerase gene into pBR322 under a lambda PL promoter and inducing expression yields a 200‑fold increase, with the enzyme comprising 20% of soluble E.

Abstract

The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.