Functional expression in insect cells, one-step, purification and characterization of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp)

Abstract

Phospholipase D (PLD) is an important enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. In this study, large amounts of a recombinant plant PLDα were secreted into the culture medium of baculovirus-infected insect cells and purified to homogeneity in the form of a fully active enzyme. The transient production of recombinant PLDα yielded a protein (rPLDαa, 88 kDa) together with a shorter form (rPLDαb, 87 kDa), which accumulated in the medium. N-Terminal amino acid sequencing of the rPLDαa and rPLDαb showed that rPLDαb resulted from proteolytic cleavage at Gly8-Ile9. Immunoblotting showed that both rPLDαa and rPLDαb are recognized by a polyclonal antibody previously raised against native soybean PLDα. One-step calcium-dependent octyl-Sepharose chromatography was used to obtain the two highly purified forms of rPLDα, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry. The N-terminal region of PLDα is homologous with the C2 domains which are present in a number of enzymes known to be involved in signal transduction and/or phospholipid metabolism. The truncated rPLDαb lacks the first acidic amino acid in its N-terminus, which is probably involved in the calcium binding site. The rPLDαb was thus easily eluted from the octyl-Sepharose column by decreasing the calcium concentration of the buffer from 50 to 30 mM, whereas, the rPLDα was eluted after chelating calcium ions with EDTA. The purified rPLDα yield reached a level of 10 mg per liter of serum-free culture medium. The availability of baculovirus-derived rPLDα constitutes a valuable source of enzyme for future crystallographic studies to determine its three-dimensional structure.