Molecular cloning, expression, and characterization of secretory phospholipase A 2 in tobacco

Phospholipase A 2 (PLA 2 ) activity was investigated in various tissues of tobacco ( Nicotiana tabacum ). PLA 2 activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA 2 activity in the flower appears to have originated from both Ca 2+ ‐dependent and‐independent PLA 2 . A cDNA clone for protein with homology to animal secretory PLA 2 (sPLA 2 ), denoted as Nt PLA 2 , was isolated from the tobacco flower. The cDNA of Nt PLA 2 encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA 2 contains 12 cysteines, a Ca 2+ binding loop, and a catalytic domain that are commonly conserved in animal sPLA 2 . The Nt PLA 2 mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA 2 was expressed as a fusion protein with thioredoxin in Escherichia coli . From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA 2 was purified by ion exchange chromatography. It was discovered that the purified Nt PLA 2 essentially requires Ca 2+ , for the enzyme activity when the activity was determined using mixed‐micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA 2 was at pH 8–10 when PC was used as a substrate.