Recombinant expression and characterization of three group IB phospholipase A2 isoforms in the red sea bream Chrysophrys major.

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. [ABSTRACT FROM AUTHOR]