不同因素对人Ⅹ型磷脂酶A2包涵体重折叠的影响

Effects on Human Group Ⅹ Phospholipase A2 Inclusion Bodies Folding

人Ⅹ型磷脂酶A2 在大肠杆菌中的表达产物完全以不溶的包涵体形式存在. 用以前适用于人胰型(IB 型) 磷脂酶A2 的稀释重折叠方法,并不能使它有效重折叠. 以初步纯化的包涵体为对象,发现溶液的温度、pH 值和蛋白质浓度对Ⅹ型磷脂酶A2 体外重折叠有很大的影响. 研究了一些小分子化合物对Ⅹ型磷脂酶A2 ,在高蛋白质浓度(1 g/L)下重折叠的影响,发现1 mol/L 的L-精氨酸能提高其重折叠效率达6倍多. L-精氨酸的结构类似物L-瓜氨酸对Ⅹ型磷脂酶A2 的重折叠有较弱的改善,而L-赖氨酸和L-精氨酸甲基酯则降低了Ⅹ型磷脂酶A2 的活性恢复. 结果表明,L-精氨酸对蛋白质重折叠的帮助作用,可能是通过精氨酸与折叠中间物的结合产生的,精氨酸的胍基和羧基都是必需的,其侧链胍基以其特殊的带电方式阻遏了重折叠过程中的积聚和分子间二硫键形成的反应.

Human group X phospholipase A(2) is a member of mammalian secretory phospholipase which belongs to phospholipase A(2) superfamily. It has been expressed in a form of inclusion bodies in E.coli. It could not retold efficiently as human pancreatic phospholipase A(2) (group I B) merely by diluting the protein unfolded in 8 mol/L of urea. The refolding reaction of human group X phospholipase A(2) in vitro was depended on temperature, pH and protein concentration. A number of additives have been tested, among which L-arginine was the most efficient effector in improving the refolding of human group X phospholipase A(2), and its structural analogs, L-citrulline, had less effect. L-lysine and L-arginine methyl ester could not improve phospholipase A(2) refolding. In addition, L-arginine inhibited the formation of aggregates and of intramolecular disulfide bonds. These results showed that both carboxyl and guanidyl residues of L-arginine were essential for improving protein refolding.