Abstract
Phospholipase D (PLD) is one of the main enzymes involved in signal transduction, vesicle trafficking and membrane metabolism processes. Here we describe the heterologous high-yield expression in the yeast Pichia pastoris, one-step purification and characterization of catalytically active PLDα from cowpea ( Vigna unguiculata L. Walp). Immunoblotting experiments showed that recombinant PLDα is recognized by a polyclonal antibody raised against native soybean PLDα. A single calcium-dependent octyl-Sepharose chromatography step was used to obtain a highly purified recombinant PLDα, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry data. From 1 L of yeast culture medium, about 8 mg of pure recombinant PLDα was obtained and the specific activity measured on phosphatidylcholine was 27 μmol/min/mg. Contrary to what was observed previously with Vigna unguiculata PLDα expressed in insect cells, no proteolytic degradation of the N-terminal calcium-dependent C2 lipid binding domain was observed here. This functional recombinant PLDα should provide a valuable tool for performing detailed studies on the molecular characterization of enzymes as well as structural studies.
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