High-level constitutive expression in Pichia pastoris and one-step purification of phospholipase D from cowpea (Vigna unguiculata L. Walp)

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 PLDalpha from cowpea (Vigna unguiculata L. Walp). Immunoblotting experiments showed that recombinant PLDalpha is recognized by a polyclonal antibody raised against native soybean PLDalpha. A single calcium-dependent octyl-Sepharose chromatography step was used to obtain a highly purified recombinant PLDalpha, 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 PLDalpha was obtained and the specific activity measured on phosphatidylcholine was 27 micromol/min/mg. Contrary to what was observed previously with Vigna unguiculata PLDalpha expressed in insect cells, no proteolytic degradation of the N-terminal calcium-dependent C2 lipid binding domain was observed here. This functional recombinant PLDalpha should provide a valuable tool for performing detailed studies on the molecular characterization of enzymes as well as structural studies.