Fig. 3: Acceptor-site specificity of PglB homologs. | Nature Communications

Fig. 3: Acceptor-site specificity of PglB homologs.

From: Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in bacteria

Fig. 3

a Immunoblot analysis of periplasmic fractions from CLM24 cells transformed with the following plasmids: pMW07-pglΔBCDEF for making GalNAc5(Glc)GlcNAc; pMLBAD encoding DmPglB, DmPglBmut, CjPglB or CjPglBmut; and pBS-scFv13-R4XQNAT encoding the scFv13-R4 with each of the 20 amino acids in the –2 position of the C-terminal sequon as indicated. Blots were probed with anti-His antibody (top panel) and hR6 serum (bottom panel). Molecular weight (MW) markers are indicated on the left. The g0 and g1 arrows indicate un- and monoglycosylated acceptor proteins, respectively. Blots are representative of biological replicates (n = 3). Schematic created with BioRender. DeLisa, M. (2025) https://BioRender.com/wesqljn. b Heatmap analysis of the relative –2 amino acid preference of CjPglB, DgPglB, and DmPglB. Relative preferences (weaker = white; stronger = dark cyan) were determined based on densitometry analysis of the glycosylation efficiency for each acceptor protein in the anti-His immunoblot (see Supplementary Figs. 5, 6 for efficiency data). c Sequence logo showing experimentally determined acceptor-site specificity of DmPglB using glycoSNAP-based library screening of YebF(N24L)-Im7XXNXT. Source data are provided as a Source Data file.

Back to article page