Fig. 1: Bioprospecting Desulfobacterota genomes for functional PglB homologs. | Nature Communications

Fig. 1: Bioprospecting Desulfobacterota genomes for functional PglB homologs.

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

Fig. 1

a Phylogenetic tree of the PglB homologs evaluated in this study. The curated list of enzymes was generated from a BLAST search using DaPglB and DgPglB as the query sequences. CjPglB and ClPglB were added for comparison. The tree was generated by the neighbor-joining method from multiple sequence alignment using Molecular Evolutionary Genetics Analysis version 11 (MEGA11) software65. b Immunoblot analysis of periplasmic fractions from CLM24 cells transformed with plasmid pMW07-pglΔBCDEF encoding genes for biosynthesis of a modified C. jejuni heptasaccharide glycan (GalNAc5(Glc)GlcNAc), plasmid pBS-scFv13-R4DQNAT encoding the scFv13-R4DQNAT acceptor protein, and a derivative of plasmid pMLBAD encoding one of the PglB homologs as indicated. The first two lanes in left and right panels of (a) and (c) were loaded with the same positive and negative control samples (marked with asterisk). Blots were probed with polyhistidine epitope tag-specific antibody (anti-His) to detect the C-terminal 6x-His tag on the acceptor protein (top panel) and hR6 serum specific for the C. jejuni heptasaccharide glycan (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. c Glycosylation efficiency was determined by densitometric analysis, with data reported as mean ± SD. Red bars correspond to positive and negative controls generated with CjPglB and CjPglBmut, respectively; blue bars correspond to samples generated with Desulfovibrio PglBs. Statistical significance was determined by unpaired two-tailed Student’s t-test with calculated p-values represented as: *p < 0.05; ***p < 0.001; ns, not significant. Actual p-values relative to CjPglB are (from left-to-right): p = 0.541, p = 0.680, p = 0.0001, p = 0.0112, p = 0.0002, and p = 0.9586. Source data are provided as a Source Data file.

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