Rapid cloning, expression and purification of a novel high-activity alkaline phosphatase with detoxification of lipopolysaccharide

文献类型: 外文期刊

第一作者: Wu, Daichao

作者: Wu, Daichao;Teng, Da;Xi, Di;Wang, Xiumin;Wang, Xiaojie;Mao, Ruoyu;Zhang, Yong;Wang, Jianhua;Wu, Daichao;Dai, Hua;Wu, Daichao;Teng, Da;Xi, Di;Wang, Xiumin;Wang, Xiaojie;Mao, Ruoyu;Zhang, Yong;Dai, Hua;Wang, Jianhua

作者机构:

关键词: Heterologous expression;Alkaline phosphatase;Saccharomyces boulardii;Lipopolysaccharide;Dephosphorylation;TNF-alpha

期刊名称:PROCESS BIOCHEMISTRY ( 影响因子:3.757; 五年影响因子:3.665 )

ISSN: 1359-5113

年卷期: 2014 年 49 卷 3 期

页码:

收录情况: SCI

摘要: Lipopolysaccharide (LPS) is a bacterial endotoxin leading to endotoxemia. Its virulence factor 'diphosphoryl lipid A' can be abolished by alkaline phosphatase (AP). A novel AP gene (without introns) was cloned from Saccharomyces boulardii ATCC MYA-796 with a GenBank accession number KF471017, and the recombinant AP (rAP) was expressed as a soluble protein in Pichia pastoris X-33 with a yield of 43.66 mg/I at the end of 120 h of induction in a shaker flask. After purification by affinity-column chromatography, the purity of rAP was over 90%. The optimal reaction conditions of rAP were pH 9.6, temperature at 60 degrees C and 2 mM Mg2+ in diethanolamine buffer, and EDTA was a potent inhibitor of rAP activity. The specific activity of rAP was 9912.01 U/mg under the optimal conditions. Furthermore, rAP showed a broad dephosphorylation activity to LPS over a broad pH range (pH 2-10) in vitro and peaked at pH 4 in Tris-HCI buffer. After LPS dephosphorylated by rAP was injected intraperitoneally into mice, the serum level of tumor necrosis factor (TNF)-alpha was significantly reduced compared to that of the LPS group (p < 0.01). These findings suggest that rAP has great potential to cure diseases caused by LPS. (C) 2014 Elsevier Ltd. All rights reserved.

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