Site-specific protein labeling using PRIME and chelation-assisted click chemistry
作者： Chayasith Uttamapinant , Mateo I Sanchez , Daniel S Liu , Jennifer Z Yao , Katharine A White
关键词:
摘要: This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase (LplA) attaches a picolyl azide (pAz) derivative 13-aa recognition sequence that has been genetically fused onto protein of interest. Proteins bearing pAz are chemoselectively derivatized alkyne-probe conjugate CuAAC second step. We describe herein optimized protocols synthesize perform labeling and achieve derivatization on live cells, fixed cells proteins. Reagent preparations, including synthesis expression LplA, take 12 d, whereas procedure for performing site-specific ligation takes 40 min-3 h.
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