Abstract
We have previously cloned three distinct isoforms of group IB phospholipase A2 (PLA2), hepatopancreas DE-1 and DE-2 PLA2 and gill G-3 PLA2, from the red sea bream Chrysophrys major and found that they are expressed in a tissue-specific manner in the red sea bream. To understand the function of three group IB PLA2 isoforms, we constructed a bacterial expression system for DE-1, DE-2 and G-3 PLA2. The cDNAs encoding for mature PLA2 were amplified by polymerase chain reaction (PCR) and subcloned in-frame with a glutathione S-transferase (GST) encoded by the vector pGEX-4T-1. The resulting plasmids were used to transform Escherichia coli BL21 (DE3) cells. Three recombinant PLA2 were expressed as a fusion protein with GST in E. coli cells by induction of Isopropyl-1-thio-β-D-galactopyranoside. The bacterial cells were lyzed with strong alkaline solution and the fusion proteins were recovered as a soluble form. The fusion proteins were purified with affinity chromatography and cleaved by trypsin. Then, the recombinant DE-1 and G-3PLA2 were purified to near homogeneity by reversed-phase high-performance liquid chromatography (HPLC), and the recombinant DE-2 PLA2 by ion exchange chromatography and reversed-phase HPLC. Enzyme properties of purified recombinant DE-1, DE-2 and G-3 PLA2 were found to be essentially the same as those of native PLA2.
