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Expression and Purification of Recombinant Vigna unguiculata Phospholipase D in Pichia pastoris for Structural Studies.

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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https://hal.univ-grenoble-alpes.fr/hal-01862139
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Soumis le : lundi 27 août 2018 - 09:01:34
Dernière modification le : mercredi 15 juillet 2020 - 13:02:04

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Yani Arhab, Renaud Rahier, Alexandre Noiriel, Mickael V Cherrier, Abdelkarim Abousalham. Expression and Purification of Recombinant Vigna unguiculata Phospholipase D in Pichia pastoris for Structural Studies.. Methods in Molecular Biology, Humana Press/Springer Imprint, 2018, 1835, pp.191-201. ⟨10.1007/978-1-4939-8672-9_10⟩. ⟨hal-01862139⟩

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