Fig. 3: Compatibility of SIMPLEx reformatting with diverse expression platforms.
From: A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans
Immunoblot analysis of the soluble (S) and insoluble (I) fractions derived from a cell-free protein synthesis (CFPS) using crude S30 extract prepared from E. coli BL21(DE3) cells; and cell-based expression using b S. cerevisiae strain SBY49 or c HEK293T cells as indicated. All three systems involved plasmids for expressing either Sx-Δ26HsST6Gal1 (Sx-ST6) or unfused Δ26HsST6Gal1 (ST6). Empty plasmid was used as a negative control (empty) in each case. Βlots were probed with anti-polyhistidine (αHis) antibody to detect GT expression, with longer exposures (αHis - long) provided to better identify protein products with low expression. An equivalent amount of total protein was loaded in each lane and confirmed by probing blots with antibodies specific for GroEL, Tubulin, and GAPDH, which are housekeeping proteins in E. coli, yeast, and mammalian cells, respectively. Results are representative of three biological replicates. Molecular weight (Mw) markers are shown on the left. Cartoon images were created with BioRender.com.
