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Protein Engineering, Vol. 13, No. 11, 811-817, November 2000
© 2000 Oxford University Press

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

Hayat El Maarouf, Frédéric Carrière, Mireille Rivière and Abdelkarim Abousalham,1

UPR 9025 du CNRS, Laboratoire de Lipolyse Enzymatique,31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France

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 PLD{alpha} 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 PLD{alpha} yielded a protein (rPLD{alpha}a, 88 kDa) together with a shorter form (rPLD{alpha}b, 87 kDa), which accumulated in the medium. N-Terminal amino acid sequencing of the rPLD{alpha}a and rPLD{alpha}b showed that rPLD{alpha}b resulted from proteolytic cleavage at Gly8–Ile9. Immunoblotting showed that both rPLD{alpha}a and rPLD{alpha}b are recognized by a polyclonal antibody previously raised against native soybean PLD{alpha}. One-step calcium-dependent octyl-Sepharose chromatography was used to obtain the two highly purified forms of rPLD{alpha}, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry. The N-terminal region of PLD{alpha} 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 rPLD{alpha}b lacks the first acidic amino acid in its N-terminus, which is probably involved in the calcium binding site. The rPLD{alpha}b was thus easily eluted from the octyl-Sepharose column by decreasing the calcium concentration of the buffer from 50 to 30 mM, whereas, the rPLD{alpha}a was eluted after chelating calcium ions with EDTA. The purified rPLD{alpha} yield reached a level of 10 mg per liter of serum-free culture medium. The availability of baculovirus-derived rPLD{alpha} constitutes a valuable source of enzyme for future crystallographic studies to determine its three-dimensional structure.


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