201. Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis.
- Author
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Le, Zhen, Li, XingZhang, Yuan, Peng, Liu, Pi, and Huang, Chunhong
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SNAKE venom , *GENE expression , *ENZYME inhibitors , *PHOSPHOLIPASE A2 , *EICOSANOIDS , *HEMORRHAGE treatment , *NEUROTOXICOLOGY - Abstract
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 PLIγ level in the serum and snake resource are not sufficiently sustainable for further research. To overcome these limitations, we constructed a His6-PLIγ pET28 fusion expression vector and transformed Escherichia coli BL21. To improve the expression of PLIγ, 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-β-d-thiogalactoside (IPTG) for 6 h to the recombinant BL21 after its OD 600 was 0.8, with continuous shaking cultivation at 190 rpm and 35 °C. Under these conditions, the amount of expressed protein could reach 57 mg/L. The His6-PLIγ was purified by nickel affinity chromatography and renatured by On-column refolding. The resulting PLIγ showed a good inhibitory effect of enzymatic activities to venom PLA 2 isolated from D. acutus . Moreover, the PLIγ had a wide anti-hemorrhage activities to D. acutus , Naja atra and Agkistrodon halys venom. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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