title: Renaturation and one step purification of the chicken GIIA secreted phospholipase A2 from inclusion bodies.
abstract: The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group IIA sPLA2, has been amplified from chicken intestine. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3mg/l of pure refolded fully active enzyme to be obtained. Recombinant expression of chicken intestinal sPLA2-IIA (ChPLA2-IIA) in Escherichia coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 10-fold preference. Indeed, we report in this work, a comparative kinetic study between the wild type and the recombinant ChPLA2-IIA, on zwitterionic head group phospholipids (DDPC) and negatively charged phospholipids (POPG) using the monomolecular film technique. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme.
authors.affiliation: Laboratoire de Biochimie et de Génie Enzymatique des Lipases, University of Sfax, ENIS Route de Soukra, BP 1173, 3038, Sfax, Tunisia.
authors.forename: Aida
authors.initials: A
authors.lastname: Karray
authors.affiliation: CNRS-Aix-Marseille Université-Enzymologie Interfaciale et Physiologie de la Lipolyse-UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
authors.forename: Sawsan
authors.initials: S
authors.lastname: Amara
authors.affiliation: CNRS-Aix-Marseille Université-Enzymologie Interfaciale et Physiologie de la Lipolyse-UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Carrière
authors.affiliation: Laboratoire de Biochimie et de Génie Enzymatique des Lipases, University of Sfax, ENIS Route de Soukra, BP 1173, 3038, Sfax, Tunisia.
authors.forename: Youssef
authors.initials: Y
authors.lastname: Gargouri
authors.affiliation: Laboratoire de Biochimie et de Génie Enzymatique des Lipases, University of Sfax, ENIS Route de Soukra, BP 1173, 3038, Sfax, Tunisia. Electronic address: sofiane_bezzine@yahoo.com.
authors.forename: Sofiane
authors.initials: S
authors.lastname: Bezzine
mesh_terms: D000818:Animals; D002645:Chickens / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D054501:Group II Phospholipases A2 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000201:enzymology* / Q000378:metabolism; D007422:Intestines / Q000201:enzymology; D008460:Meat; D016297:Mutagenesis, Site-Directed
chemical_list: D018076:DNA, Complementary; D054501:Group II Phospholipases A2
keywords: Gene expression; In vitro refolding; Phospholipase A2