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 II sPLA2, has been amplified. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3 mg per litre of pure refolded fully active enzyme to be obtained. Recombinant expression of 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 15-fold preference. 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, Tunisia. karrayaida_biotech@yahoo.fr
authors.forename: Aida
authors.initials: A
authors.lastname: Karray
authors.forename: Madiha
authors.initials: M
authors.lastname: Bou Ali
authors.forename: Sawsan
authors.initials: S
authors.lastname: Amara
authors.forename: Frédéric
authors.initials: F
authors.lastname: Carrière
authors.forename: Youssef
authors.initials: Y
authors.lastname: Gargouri
authors.forename: Sofiane
authors.initials: S
authors.lastname: Bezzine
keywords: A2; ChPLA2-IIAchicken intestinal PLA2; Gene expression; In vitro refolding; Phospholipase A2; sPLA2; secreted phospholipase