A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins

Recombinant immunotoxins (rITs) are highly specific anti-tumor agents compo sed of monoclonal antibody fragments or other specific carriers coupled to plant or bacterial toxins. A major problem in the purification of rITs is t he low periplasmic yield in currently available expression systems. Thus, t he aim of this study was the development of a new bacterial expression syst em for high-level production of rITs. We constructed a series of pET-based vectors for pelB-directed periplasmic secretion or cytoplasmic production u nder the control of the T7lac promoter. Expression in Escherichia coli BL21 (DE3)pLysS allowed a tightly regulated isopropyl beta-d-thiogalactopyranosi de (IPTG) induction of protein synthesis. An enterokinase-cleavable poly-hi stidine cluster was introduced into this setup for purification by affinity chromatography. A major modification resulted from the insertion of a spec ifically designed multiple cloning site. It contains only rare restriction enzyme recognition sites used for cloning of immunoglobulin variable region genes, as well as unique SfiI and NotI restriction sites for directed inse rtion of single-chain variable fragments (scFv) available from established bacteriophage systems. For this purpose, we deleted two naturally occurring internal SfiI consensus sites in a deletion mutant of Pseudomonas aerugino sa exotoxin A (ETA'). Each single structural element of the new vector (pro moter, leader sequence, purification tag, scFv sequence, selectable marker, and toxin gene) was flanked by unique restriction sites allowing simple di rectional substitution. The fidelity of IPTG induction and high-level expre ssion were demonstrated using an anti-CD30 scFv (Ki-4) fused to ETA'. These data confirm a bacterial vector system especially designed for efficient p eriplasmic expression of ETA'-based fusion toxins. (C) 1999 Elsevier Scienc e B.V. All rights reserved.