High-yield expression, purification, and characterization of the recombinant diisopropylfluorophosphatase from Loligo vulgaris

Organophosphate degrading enzymes are of great interest in light of their a bility to detoxify chemical warfare agents. The diisopropylfluorophosphatas e (DFPase) from Loligo vulgaris is characterized by its high stability and broad substrate specifity. Here we report the production of large amounts o f active, recombinant DFPase using an Escherichia coli expression system. T he enzyme was purified to homogeneity using a combination of immobilized me tal affinity and ion exchange chromatography. CD-spectroscopy indicates a w ell folded protein with a high amount of beta -sheet structure. Limited pro teolysis was used to gain a deeper insight into the structural organization of the protein. DFPase possesses an internal protease-sensitive region loc ated between amino acids 146 and 149. The two proteolytic fragments remain as a tight complex retaining a DFPase activity comparable to the intact enz yme. Overexpression clones for each fragment were constructed with the expr ession resulting in the formation of inclusion bodies. Upon isolation and r efolding active protein is only formed when both fragments are present. Thu s, the two proteolytic fragments are probably part of a stable single-domai n protein with multiple contacts between them and only one protease accessi ble surface loop. (C) 2001 Academic Press.