BACTERIAL EXPRESSION AND CHARACTERIZATION OF HUMAN PANCREATIC PHOSPHOLIPASE A(2)

Mammalian pancreatic phospholipases A(2) (PLA(2)) have recently been i mplicated in cell surface receptor-mediated inflammation. As a first s tep toward understanding how human pancreatic PLA(2) (hp-PLA(2)) inter acts with membranes and other biological targets including cell-surfac e receptors, we constructed its bacterial expression vector which can be used for the mutagenesis and protein over-expression. The expressio n vector (pSH-hp) was constructed using a synthetic hp-PLA(2) gene who se transcription is controlled by T7 promoter. hp-PLA(2) was expressed as a mature protein in high concentration in Escherichia coli cells a nd formed inclusion body. The solubilization of inclusion body protein followed by the refolding and purification produced ca. 5 mg of pure protein from one liter of growth medium. Kinetic studies of recombinan t human, bovine and porcine pancreatic PLA(2)s using polymerized mixed Liposomes and micelles as substrates showed that despite their highly homologous structures these mammalian pancreatic PLA(2)s have distinc t phospholipid head group specificity and different activity toward va rious lipid substrates.