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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References
Jenkins GM, Frohman MA (2005) Phospholipase D: a lipid centric review. Cell Mol Life Sci 62(19-20):2305–2316. https://doi.org/10.1007/s00018-005-5195-z
Selvy PE, Lavieri RR, Lindsley CW, Brown HA (2011) Phospholipase D: enzymology, functionality, and chemical modulation. Chem Rev 111(10):6064–6119. https://doi.org/10.1021/cr200296t
Ulbrich-Hofmann R, Lerchner A, Oblozinsky M, Bezakova L (2005) Phospholipase D and its application in biocatalysis. Biotechnol Lett 27(8):535–544. https://doi.org/10.1007/s10529-005-3251-2
Zheng L, Krishnamoorthi R, Zolkiewski M, Wang X (2000) Distinct Ca2+ binding properties of novel C2 domains of plant phospholipase dalpha and beta. J Biol Chem 275(26):19700–19706. https://doi.org/10.1074/jbc.M001945200
Zheng L, Shan J, Krishnamoorthi R, Wang X (2002) Activation of plant phospholipase Dbeta by phosphatidylinositol 4,5-bisphosphate: characterization of binding site and mode of action. Biochemistry 41(14):4546–4553
Hanahan DJ, Chaikoff IL (1947) The phosphorus-containing lipides of the carrot. J Biol Chem 168(1):233–240
Abousalham A, Riviere M, Teissere M, Verger R (1993) Improved purification and biochemical characterization of phospholipase D from cabbage. Biochim Biophys Acta 1158(1):1–7. https://doi.org/10.1016/0304-4165(93)90088
Wang X, Dyer JH, Zheng L (1993) Purification and immunological analysis of phospholipase D from castor bean endosperm. Arch Biochem Biophys 306(2):486–494
Abousalham A, Teissere M, Gardies AM, Verger R, Noat G (1995) Phospholipase D from soybean (Glycine max L.) suspension-cultured cells: purification, structural and enzymatic properties. Plant Cell Physiol 36(6):989–996
Abdelkafi S, Abousalham A (2011) Kinetic study of sunflower phospholipase Dalpha: interactions with micellar substrate, detergents and metals. Plant Physiol Biochem 49(7):752–757. https://doi.org/10.1016/j.plaphy.2011.02.002
Khatoon H, Mansfeld J, Schierhorn A, Ulbrich-Hofmann R (2015) Purification, sequencing and characterization of phospholipase D from Indian mustard seeds. Phytochemistry 117:65–75. https://doi.org/10.1016/j.phytochem.2015.05.022
Zhao J, Wang X (2004) Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. J Biol Chem 279(3):1794–1800. https://doi.org/10.1074/jbc.M309529200
Wang X, Xu L, Zheng L (1994) Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L. J Biol Chem 269(32):20312–20317
Ueki J, Morioka S, Komari T, Kumashiro T (1995) Purification and characterization of phospholipase D (PLD) from rice (Oryza sativa L.) and cloning of cDNA for PLD from rice and maize (Zea mays L.). Plant Cell Physiol 36(5):903–914
Schäffner I, Rücknagel K-P, Mansfeld J, Ulbrich-Hofmann R (2002) Genomic structure, cloning and expression of two phospholipase D isoenzymes from white cabbage. Eur J Lipid Sci Technol 104(2):79–87
Dyer JH, Zheng L, Wang X (1995) Cloning and nucleotide sequence of a cDNA encoding phospholipase D from arabidopsis. Plant Physiol 109:1497
El Maarouf H, Carriere F, Riviere M, Abousalham A (2000) Functional expression in insect cells, one-step purification and characterization of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp). Protein Eng 13(11):811–817
Ben Ali Y, Carriere F, Abousalham A (2007) High-level constitutive expression in Pichia pastoris and one-step purification of phospholipase D from cowpea (Vigna unguiculata L. Walp). Protein Expr Purif 51(2):162–169. https://doi.org/10.1016/j.pep.2006.07.018
Rahier R, Noiriel A, Abousalham A (2016) Functional characterization of the N-terminal C2 domain from Arabidopsis thaliana phospholipase Dalpha and Dbeta. Biomed Res Int 2016:2721719. https://doi.org/10.1155/2016/2721719
Lambrecht R, Ulbrich-Hofmann R (1992) A facile purification procedure of phospholipase D from cabbage and its characterization. Biol Chem Hoppe Seyler 373(2):81–88
Leiros I, Secundo F, Zambonelli C, Servi S, Hough E (2000) The first crystal structure of a phospholipase D. Structure 8(6):655–667
Ponting CP, Kerr ID (1996) A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues. Protein Sci 5(5):914–922. https://doi.org/10.1002/pro.5560050513
Ullah A, de Giuseppe PO, Murakami MT, Trevisan-Silva D, Wille AC, Chaves-Moreira D, Gremski LH, da Silveira RB, Sennf-Ribeiro A, Chaim OM, Veiga SS, Arni RK (2011) Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom. Acta Crystallogr Sect F Struct Biol Cryst Commun 67(Pt 2):234–236. https://doi.org/10.1107/S1744309110050931
Abergel C, Abousalham A, Chenivesse S, Riviere M, Moustacas-Gardies AM, Verger R (2001) Crystallization and preliminary crystallographic study of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp). Acta Crystallogr D Biol Crystallogr 57(Pt 2):320–322
Stumpe S, Konig S, Ulbrich-Hofmann R (2007) Insights into the structure of plant alpha-type phospholipase D. FEBS J 274(10):2630–2640. https://doi.org/10.1111/j.1742-4658.2007.05798.x
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd. edn. Cold Spring Harbor Laboratory Press, New York
Takrama JF, Taylor KE (1991) A continuous spectrophotometric method for monitoring phospholipase D-catalyzed reactions of physiological substrates. J Biochem Biophys Methods 23(3):217–226
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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