Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis
Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis
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天然蛇毒磷脂酶 A(2) 抑制剂原核表达的正交优化
DOI：
10.1016/j.toxicon.2015.10.018
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发表时间：
2015-12-15
期刊：
影响因子：
2.8
通讯作者：
Huang, Chunhong
中科院分区：
文献类型：
--
作者：
Le, Zhen;Li, XingZhang;Huang, Chunhong
磷脂酶A（2）（PLA（2））是一种钙依赖性酶，参与炎症过程，例如从膜池释放游离花生四烯酸以生物合成类花生酸。已知蛇毒含有PLA（2）s（svPLA（2）s），其表现出多种药理学作用，包括神经毒性、心脏毒性、肌肉毒性和出血。因此，抑制svPLA（2）将有利于成功地进行毒液蛰入治疗。从中国特有的无毒蛇类环纹华蛇（Sinonatrix annularis）血清中提取了一种γ型磷脂酶I（PLA（2）抑制剂）。这表明尖吻蝮蛇PLA具有很强的抑制作用（2），然而，血清和蛇资源中的PLI γ水平不足以持续进行进一步研究。为了克服这些限制，我们构建了His 6-PLI γ pET 28融合表达载体并转化大肠杆菌BLI γ。为了提高PLI γ的表达，进行正交实验[L-16（4）（5）]以优化诱导参数。确定了重组菌的最佳诱导条件为：0.4 mM异丙基-β-d-硫代半乳糖苷（IPTG）诱导表达，待重组菌OD 600达到0.8后，在190 rpm、35 ℃条件下连续振荡培养6 h。在此条件下，表达量可达57 mg/L。镍亲和层析纯化His 6-PLI γ，柱上复性。PLI γ对D. acutus PLI γ对D.尖吻蝮蛇毒、眼镜蛇毒和蝮蛇毒。(C)2015爱思唯尔有限公司版权所有。
Phospholipase A(2) (PLA(2)) is a calcium-dependent enzyme that is involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Snake venom are known containing PLA(2)s (svPLA(2)s) which exhibit a wide variety of pharmacological effects including neurotoxicity, cardiotoxicity, myotoxicity and hemorrhage. Therefore, inhibition of svPLA(2) would be advantageous to successful envenomation treatment. A gamma type PLI (PLA(2) inhibitor) has been extracted from the serum of Sinonatrix annularis, a non-venomous snake indigenous to China. This showed strong inhibition of Deinagkistrrodon acutus PLA(2), however, the PLI gamma level in the serum and snake resource are not sufficiently sustainable for further research. To overcome these limitations, we constructed a His6-PLI gamma pET28 fusion expression vector and transformed Escherichia coli BLI gamma. To improve the expression of PLI gamma, an orthogonal experiment [L-16(4)(5)] was performed to optimize induction parameters. The optimized condition was determined to be: induction by 0.4 mM isopropyl-beta-d-thiogalactoside (IPTG) for 6 h to the recombinant BL21 after its OD600 was 0.8, with continuous shaking cultivation at 190 rpm and 35 degrees C. Under these conditions, the amount of expressed protein could reach 57 mg/L The His6-PLI gamma was purified by nickel affinity chromatography and renatured by On-column refolding. The resulting PLI gamma showed a good inhibitory effect of enzymatic activities to venom PLA(2) isolated from D. acutus. Moreover, the PLI gamma had a wide anti-hemorrhage activities to D. acutus, Naja atra and Agkistrodon halys venom. (C) 2015 Elsevier Ltd. All rights reserved.