Abstract
The production of pure enzymes in high quantities is a proven strategy to study the catalytic mechanism as well as the solving of structure at the atomic scale for therapeutic or industrial purposes. Phospholipase D (PLD, EC 3.1.4.4) is found in a wide majority of living organisms and has been shown to be involved in signal transduction, vesicle trafficking, and membrane metabolism processes. Located at the membrane-cytoplasm interface, plant PLDs are soluble but also bear an evident hydrophobic aspect making challenging its expression and its purification in large quantity. So far there is no high-resolution three-dimensional structure for a eukaryotic PLD. The protocols herein describe the cloning of the eukaryotic recombinant PLDα of Vigna unguiculata (cowpea) into the yeast expression system Pichia pastoris and its two-step purification process. This allowed us to purify to homogeneity hundreds of micrograms of highly pure protein to conduct in fine structural studies.
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Acknowledgments
The French “Ministère de l’Enseignement supérieur et de la Recherche” is gratefully acknowledged for the funding of Yani Arhab and Renaud Rahier.
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Arhab, Y., Rahier, R., Noiriel, A., Cherrier, M.V., Abousalham, A. (2018). Expression and Purification of Recombinant Vigna unguiculata Phospholipase D in Pichia pastoris for Structural Studies. In: Sandoval, G. (eds) Lipases and Phospholipases. Methods in Molecular Biology, vol 1835. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8672-9_10
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DOI: https://doi.org/10.1007/978-1-4939-8672-9_10
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