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Autodisplay of human PIP5K1α lipid kinase on Escherichia coli and inhibitor testing
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2021-02-01 , DOI: 10.1016/j.enzmictec.2020.109717
Katja Strätker 1 , Samer Haidar 2 , Mariam Dubiel 1 , Ana Estévez-Braun 3 , Joachim Jose 1
Affiliation  

The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) plays a major role in the PI3K/AKT/mTOR signaling pathway. As it has been shown before that hPIP5K1α is involved in the development of different types of cancer in particular prostate cancer, inhibitors of the enzyme might be a new option for the treatment of this disease. Here we report on the expression of hPIP5K1α on the surface of E. coli using Autodisplay. Autodisplay is defined as the surface display of a recombinant protein on a gramnegative bacterium by the autotransporter secretion pathway. After verification of surface expression, enzyme activity of whole cells displaying hPIP5K1α was determined by a capillary electrophoresis based assay. When using cells at an OD578 of 2.5, the artificial substrate phosphatidylinositol4-phosphate (PI(4)P) fluorescein was converted by a rate of 10.7 ± 0.2 fmol/min. Using this substrate inhibition of three pyranobenzoquinone type compounds was tested. The most active compound was 4-(2-amino-3-cyano-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-chromen-4-yl) benzoic acid with an IC50 value of 8.6 μM. Because until now, all attempts to purify hPIP5K1α failed, we suggest the use of whole cells of E. coli displaying the enzyme as a convenient tool for inhibitor identification.

中文翻译:

人 PIP5K1α 脂质激酶在大肠杆菌上的自动展示和抑制剂测试

人磷脂酰肌醇 4-磷酸 5-激酶 I 型 (hPIP5K1α) 在 PI3K/AKT/mTOR 信号通路中起主要作用。正如之前所表明的,hPIP5K1α 参与了不同类型癌症的发展,尤其是前列腺癌,该酶的抑制剂可能是治疗这种疾病的新选择。在这里,我们使用 Autodisplay 报告了 hPIP5K1α 在大肠杆菌表面的表达。自展示被定义为通过自转运蛋白分泌途径在革兰氏阴性细菌上的重组蛋白的表面展示。在验证表面表达后,显示 hPIP5K1α 的全细胞的酶活性通过基于毛细管电泳的测定确定。当使用 OD578 为 2.5 的细胞时,人工底物磷脂酰肌醇 4-磷酸 (PI(4)P) 荧光素以 10.7 ± 0.2 fmol/min 的速率转化。测试了使用这种底物对三种吡喃苯醌类化合物的抑制作用。最活跃的化合物是 4-(2-amino-3-cyano-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-chromen-4-yl) 苯甲酸,IC50 值8.6 μM。因为到目前为止,所有纯化 hPIP5K1α 的尝试都失败了,我们建议使用显示该酶的大肠杆菌全细胞作为抑制剂鉴定的便捷工具。
更新日期:2021-02-01
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