Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli

Abstract

A generic vector, pASK75, was developed for the synthesis of foreign proteins in Escherichia coli under transcriptional control of the tetA promoter/operator. Tight regulation was achieved by placing the structural gene for the tet represser, as a transcriptional fusion, downstream from the β-lactamase-encoding gene (bla) on the same plasmid. Strong expression of the foreign gene was conveniently induced by adding anhydrotetracycline at a low concentration. Using the production of a recombinant murine immunoglobulin Fab fragment as an example, the system was shown to function independently of the host-strain background and to be extremely well repressed in the absence of the inducer. Thus, it represents an economic and independent alternative to IPTG-inducible promoter constructs. Additional features of pASK75 include a signal sequence and a multiple cloning site followed by a region encoding the Strep tag affinity peptide to facilitate purification of a bacterially produced protein.