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 composed of monoclonal antibody fragments or other specific carriers coupled to plant or bacterial toxins. A major problem in the purification of rITs is the low periplasmic yield in currently available expression systems. Thus, the aim of this study was the development of a new bacterial expression system for high-level production of rITs. We constructed a series of pET-based vectors for pelB-directed periplasmic secretion or cytoplasmic production under the control of the T7lac promoter. Expression in Escherichia coli BL21(DE3)pLysS allowed a tightly regulated isopropyl beta-d-thiogalactopyranoside (IPTG) induction of protein synthesis. An enterokinase-cleavable poly-histidine cluster was introduced into this setup for purification by affinity chromatography. A major modification resulted from the insertion of a specifically 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 insertion 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 aeruginosa exotoxin A (ETA'). Each single structural element of the new vector (promoter, leader sequence, purification tag, scFv sequence, selectable marker, and toxin gene) was flanked by unique restriction sites allowing simple directional substitution. The fidelity of IPTG induction and high-level expression were demonstrated using an anti-CD30 scFv (Ki-4) fused to ETA'. These data confirm a bacterial vector system especially designed for efficient periplasmic expression of ETA'-based fusion toxins.
Matthey
B
B
Department I of Internal Medicine at the University Hospital of Cologne, Laboratory of Immunotherapy, Joseph-Stelzmann-Str. 9, 50931, Cologne, Germany.
Engert
A
A
Klimka
A
A
Diehl
V
V
Barth
S
S
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000235:genetics; D005098:Exotoxins / Q000235:genetics*; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology; D000922:Immunotoxins / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D017730:Ki-1 Antigen / Q000235:genetics; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004262:DNA Restriction Enzymes; D005098:Exotoxins; D007141:Immunoglobulin Fc Fragments; D000922:Immunotoxins; D007544:Isopropyl Thiogalactoside; D017730:Ki-1 Antigen; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A