Abdullah K M, Leger S, Perrier H, Cromlish W A, Kennedy B, Gresser M
Department of Biochemistry, Merck Frosst Center for Therapeutic Research, Point Claire-Dorval, Quebec, Canada.
Protein Expr Purif. 1995 Jun;6(3):291-7. doi: 10.1006/prep.1995.1038.
Cytosolic phospholipase A2 (cPLA2) plays a key role in the production of proinflammatory lipid mediators such as prostaglandins, thromboxane, and leukotrienes. cPLA2, an arachidonic acid-selective, 85-kDa protein has been purified, cloned, and partially characterized from a number of tissues. However, the purification schemes previously published by several groups are lengthy, involving several chromatographic steps and resulting in low yields of enzyme. Here we report the preparation of a novel affinity column (Affi-656) by immobilizing a competitive inhibitor of cPLA2, and a single-step purification of this enzyme. This column selectively retains cPLA2 activity from the cytosolic fractions of Sf9 cells infected with recombinant baculovirus which is eluted by a gradient of CHAPS in the elution buffer. Purification of cPLA2 to homogeneity can thus be accomplished in a single step. Moreover, mutant cPLA2 (Ser505/Ala505) which is no longer phosphorylated at Ser505 is also retained on Affi-656; mutation at this residue does not disrupt its binding to the affinity column. To our knowledge, this is the first report of cPLA2 affinity purification. Affi-656 is a convenient, reproducible, and high-capacity affinity column, and is a valuable tool for rapid purification of cPLA2 in large quantities.
胞质磷脂酶A2（cPLA2）在前列腺素、血栓素和白三烯等促炎脂质介质的产生中起关键作用。cPLA2是一种对花生四烯酸具有选择性的85 kDa蛋白，已从多种组织中纯化、克隆并进行了部分特性鉴定。然而，此前几个研究小组发表的纯化方案冗长，涉及多个色谱步骤，且酶产量低。在此，我们报告了一种新型亲和柱（Affi - 656）的制备方法，该亲和柱通过固定cPLA2的竞争性抑制剂来实现cPLA2的一步纯化。此柱能从感染重组杆状病毒的Sf9细胞的胞质组分中选择性保留cPLA2活性，在洗脱缓冲液中用CHAPS梯度洗脱。因此，cPLA2可一步纯化至同质。此外，在Ser505位点不再磷酸化的突变型cPLA2（Ser505/Ala505）也能保留在Affi - 656上；该位点的突变并不破坏其与亲和柱的结合。据我们所知，这是cPLA2亲和纯化的首次报道。Affi - 656是一种方便、可重复且高容量的亲和柱，是快速大量纯化cPLA2的宝贵工具。
