A novel expression vector for high-level synthesis and secretion of foreign proteins in Escherichia coli: overproduction of bovine pancreatic phospholipase A 2

Abstract

A novel expression plasmid (pTO-N) has been constructed that allows for the production of large quantities of foreign proteins (or fragments thereof) in an unfused state. The vector has a strong and tightly regulated T7 10 promoter and the ompA Shine-Dalgarno (SD) sequence, followed by the ompA sequence and a cloning linker region. The mRNAs produced by the vector are protected by secondary structures at both ends of the mRNAs. The OmpA signal peptide directed the synthesized proteins into the periplasmic space of Escherichia coli. Phospholipase A 2 and prophospholipase A 2 from bovine pancreas have been produced to a high level by using this expression vector. One additional feature, which is essential for the stable maintenance of the plasmid in the E. coli expression host, BL21(DE3)[pLysS], is the shortened distance between the 5′ secondary structure sequence (immediately following the gene 10 promoter) and the SD sequence. This vector could be particularly useful for synthesis of toxins in E. coli.