Purification and gene cloning of a novel antibacterial phospholipase A2 from the sponge Agelas clathroides in Kapoposang Island Indonesia terrestrial

摘要

A new phospholipase A2 enzyme (PLA2) has been purified from the sponge of Agelas clathroides by using ammonium sulphate fractionation, column chromatography and reversed-phase HPLC. It behaves as a single-band on SDS-PAGE with molecular weight of 39 kDa. Based on amino acids partial sequence, we cloned and sequenced cDNA encoding PLA2. It consists of 474 nucleotides encoding 157 amino acid residues including a putative initiation Met. To obtain it in large amounts, the coding sequence of PLA2 was cloned into pGEX-2TK vector and expressed as a PLA2 fusion protein inudEscherichia coli BL21 strain. The soluble fusion protein collected from the supernatant of the cell lysate with induction by 50 mM isopropyl-??-D-thiogalactopyranoside (IPTG) was purified in a single-step on glutathione agarose bead chromatography. The purified native PLA2 protein and recombinant PLA2 fusion protein were determinated for novel antibacterial activity. Recombinant PLA2 fusion protein exhibited a similar antibacterial activity to the native PLA2. The recombinant PLA2 had stronger antibacterial activity toward Salmonella typhy and Staphylococus aureus (G+) with the inhibition zone diameters of 2.0 times higher than that toward Echerichia coli and Vibrio cholerae (G-). These works might provide a significant foundation for following research on the antibacterial action of PLA2 protein from marine sponges.