Renaturation and one step purification of the chicken GIIA secreted phospholipase A2 from inclusion bodies

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• 作者：Aida Karray ; Madiha Bou Ali ; Sawsan Amara ; Fr¨¦d¨¦ric Carri¨¨re ; Youssef Gargouri ; Sofiane Bezzine
• 关键词：sPLA2 ; secreted phospholipase ; A2 ; ChPLA2-IIAchicken intestinal PLA2
• 刊名：International Journal of Biological Macromolecules
• 出版年：2013
• 出版时间：September, 2013
• 年：2013
• 卷：60
• 期：Complete
• 页码：28-32
• 全文大小：1067 K

文摘

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 Ca²⁺ 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.