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 PLD alpha from cowpea (Vigna unguiculata L. Walp). Immunoblotting experiments showed that recombinant PLD alpha is recognized by a polyclonal antibody raised against native soybean PLD alpha. A single calcium-dependent octyl-Sepharose chromatography step was used to obtain a highly purified recombinant PLD alpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry data. From 1L of yeast culture medium, about 8 mg of pure recombinant PLD alpha was obtained and the specific activity measured on phosphatidylcholine was 27 mu mol/min/mg. Contrary to what was observed previously with Vigna unguiculata PLD alpha expressed in insect cells, no proteolytic degradation of the N-terminal calcium-dependent C2 lipid binding domain was observed here. This functional recombinant PLD alpha should provide a valuable tool for performing detailed studies on the molecular characterization of enzymes as well as structural studies. (c) 2006 Elsevier Inc. All rights reserved.