文章基本信息

标题：A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding
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作者：Carl W. White ; Laura E. Kilpatrick ; Kevin D.G. Pfleger 等
期刊名称：iScience
印刷版ISSN：2589-0042
出版年度：2021
卷号：24
期号：1
页码：1-18
DOI：10.1016/j.isci.2020.102011
语种：English
出版社：Elsevier
摘要：SummarySecreted chemokines are critical mediators of cellular communication that elicit intracellular signaling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome editing was used to tag the chemokine CXCL12 with the nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analyzed using BRET. These live cell approaches combine the sensitivity of nanoluciferase with CRISPR/Cas9 genome editing to detect, quantify, and monitor binding of low levels of native secreted proteins in real time.Graphical abstractDisplay OmittedHighlights•Quantification of endogenous CXCL12 secretion using CRISPR/Cas9 genome-editing•A NanoBRET approach to investigate CXCL12-receptor binding•Detection of CXCL12-GAG interactions using nanoluciferase complementationTechniques in Genetics; Molecular Interaction; Biotechnology