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. Author manuscript; available in PMC: 2022 Nov 19.
Published in final edited form as: ACS Chem Biol. 2021 Nov 2;16(11):2690–2701. doi: 10.1021/acschembio.1c00498

Figure 3. Functional analysis of PglI GTases in the heterologous E. coli glycosylation system.

Figure 3.

GTase-activities of GEMS PglIs in the E. coli 3-plasmid complementation system were analysed in whole cell lysates of E. coli CLM24 by western blotting; 5 μg of total protein were loaded in each lane. His-tag specific antibodies (upper panel) detect the glycosylation status of the glycan acceptor protein CmeA-His6; N-glycan-specific antibodies (R1, lower panel) detect the full-length Cj-N-glycan on CmeA-His6 that is only formed upon expression of an active PglI protein. Gene/plasmid combinations present in the underlying strains are indicated on top of each lane. Non, -mono, -and di-glycosylated CmeA-His6 proteins are indicated by 0N, 1N, and 2N, respectively. Molecular weight markers (Mw) in kDa are indicated on the left. Top-to-bottom scans are provided in the Figure S7.