Chapter title |
Expression and Purification of Recombinant Vigna unguiculata Phospholipase D in Pichia pastoris for Structural Studies
|
---|---|
Chapter number | 10 |
Book title |
Lipases and Phospholipases
|
Published in |
Methods in molecular biology, August 2018
|
DOI | 10.1007/978-1-4939-8672-9_10 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8671-2, 978-1-4939-8672-9
|
Authors |
Yani Arhab, Renaud Rahier, Alexandre Noiriel, Mickael V. Cherrier, Abdelkarim Abousalham, Arhab, Yani, Rahier, Renaud, Noiriel, Alexandre, Cherrier, Mickael V., Abousalham, Abdelkarim |
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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