化学
酶
熊果苷
离解常数
免疫印迹
配体(生物化学)
生物化学
酶分析
亲和层析
分子生物学
色谱法
受体
生物
基因
作者
Yuncong Yuan,Xiao-Bo Bai,Yiming Liu,Xiaonan Tang,Hao Yuan,Xun Liao
标识
DOI:10.1016/j.aca.2021.338359
摘要
Ligand fishing for screening of enzyme inhibitors from complex chemical systems using baits prepared by cell surface display of the enzyme is herein demonstrated for the first time. Tyrosine phosphatase 1B (PTP1B), used as a model enzyme in this work, is displayed on the surface of E. coli cells by using ice nucleation protein (INP) as the anchoring motif. Infusion of PTP1B is characterized by western blot, immunofluorescence, proteinase K accessibility, and enzyme activity assays. Surface displayed PTP1B exhibits a maximum of 5.62 ± 0.251 U/OD600 enzymatic activity and a better stability compared with free enzyme. PTP1B displayed cells are used as solid-phase extraction adsorbent in combination with HPLC-MS to screen the inhibitors from the extracts of Rhodiola rosea, a traditional Chinese medicinal plant. Among many well-known active ingredients only arbutin is fished out with an IC50 value of 20.5 ± 0.873 μM, showing the inhibitor screening is highly selective. Furthermore, the equilibrium dissociation constant (KD) of the complex of arbutin and PTP1B was determined to be 79.6 μM by localized surface plasma resonance (LSPR) assay. The proposed ligand fishing technique using recombinant cells as baits opens a new avenue for screening of active compounds from natural products with accuracy and specificity.
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