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Author:
Date:
2021-02-05
[Abstract] Previous expression/purification strategies for cytosolic phospholipase A2α C2-domain in Escherichia coli have relied on refolded protein recovered from inclusion bodies and sometimes containing C-terminal Cys139Ala and Cys141Ser substitutions to eliminate potential refolding complications induced by Cys residues. The protocol presented herein describes an effective method for the expression of cytosolic phospholipase A2α C2-domain in soluble form in E. coli and subsequent purification to homogeneity. This protocol, which utilizes a cleavable 6xHis-SUMO tag, has recently been used to gain insights into the structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2α (Hirano et al., 2019) ...
[摘要] [摘要]胞质磷脂酶A上一页表达/纯化策略2 α在C2结构域Ê scherichia大肠杆菌一直依赖于重折叠蛋白从包涵体回收和有时含有C末端Cys139Ala和Cys141Ser取代，以消除由感应电势再折叠的并发症的Cys残基。本文所介绍的协议描述了用于胞浆型磷脂酶A的表达的有效方法2 αC2结构域以可溶形式在大肠杆菌和随后的纯化至均一。此协议，它利用可切割的6xHis-SUMO标记，最近被用于洞察磷脂的结构基础-通过胞浆型磷脂酶A的C2结构域胆碱识别2 α（平野。等人，2019 ）
[背景技术]磷脂酶A 2 （PLA 2）是一个多样化酶超家族的成员水解的SN -2酰基酯键的磷酸甘油酯（史密斯，1989;丹尼斯。等人，2011; Mouchlis和Dennis，2019 ）。胞质PLA 2 α（与cPLA 2 α），第IV族哺乳动物PLA2家族成员，优选从在细胞内的Ca磷酸甘油酯释放花生四烯酸2+ -浓度依赖性（克拉克等人清水;，1991等人，2006; Leslie等，2010; Vasquez等，2018; Astudillo等，2019 ）。花生四烯酸通过与cPLA产生2 α用作促炎症类花生酸的前体，包括某些前列腺素和白三烯。因此，与cPLA 2 ...
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