1. Fig. 2 Scheme for the product entrapment strategy and the established cell-based fucosylation assay for FACS screening.
Scheme for the product entrapment strategy and the established cell-based fucosylation assay for FACS screening. (A) Two kinds of fluorescently labeled LacNAc derivatives (1 and 2) were designed and synthesized for cell entrapment analysis. (B) Fluorescently labeled acceptor substrates are transported into the cell via LacY; fucose enters into the cell via a fucosyl transporter (FucP) and was converted into GDP-fucose donor substrate by GDP-fucose synthase (FKP). After incubation and washing, E. coli cells expressing fucosylation enzymes accumulate fluorescent trisaccharide enzyme products, as the LacY transport rate for such products is significantly reduced compared to their disaccharide substrate form. Thus, the fluorescence intensity accumulation inside cells carries information about the catalytic activity of the fucosylation enzymes being assayed/screened. These cells with FutA activity can be further isolated using FACS. (C) Visualization of fluorescence entrapment within FutA(+) and FutA(−) cells under an ultraviolet light. (D) Flow cytometry profiles of FutA(+) and FutA(−) cell fluorescence after 30-min incubation with 1.5 mM fucose, 0.5 mM bodipy-LacNAc, and coumarin-LacNAc, followed by a washing step. Green and blue signals represent cells retaining bodipy and coumarin fluorescently labeled oligosaccharides, respectively.
Yumeng Tan et al. Sci Adv 2019;5:eaaw8451
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