Fig. 5
One-pot CFGpS using extracts selectively enriched with OSTs and LLOs. a Western blot analysis of scFv13-R4DQNAT or sfGFP217-DQNAT produced by crude CLM24 extract selectively enriched with (i) CjPglB from heterologous overexpression from pSF-CjPglB and (ii) CjLLOs from heterologous overexpression from pMW07-pglΔB. Reactions were primed with plasmid pJL1-scFv13-R4DQNAT or pJL1-sfGFP217-DQNAT. b Ribbon representation of human erythropoietin (PDB code 1BUY) with α-helixes and flexible loops colored in red and green, respectively. Glycosylation sites modeled by mutating the native sequons at N24 (22-AENIT-26), N38 (36-NENIT-40), or N83 (81-LVNSS-85) to DQNAT, with asparagine residues in each sequon colored in blue. Image prepared using UCSF Chimera package.68 Glycoengineered hEPO variants in which the native sequons at N24 (22-AENIT-26), N38 (36-NENIT-40), or N83 (81-LVNSS-85) were individually mutated to an optimal bacterial sequon, DQNAT (shown in blue). Western blot analysis of hEPO glycovariants produced by crude CLM24 extract selectively enriched with (i) CjPglB from heterologous overexpression from pSF-CjPglB and (ii) CjLLOs from heterologous overexpression from pMW07-pglΔB. Reactions were primed with plasmid pJL1-hEPO22-DQNAT-26 (N24), pJL1-hEPO36-DQNAT-40 (N38), or pJL1-hEPO81-DQNAT-85 (N83) as indicated. All control reactions (lane 1 in each panel) were performed using CjLLO-enriched extracts that lacked CjPglB. Blots were probed with anti-hexa-histidine antibody (anti-His) to detect the acceptor proteins or hR6 serum (anti-glycan) to detect the N-glycan. Arrows denote aglycosylated (g0) and singly glycosylated (g1) forms of the protein targets. Asterisks denote bands corresponding to non-specific serum antibody binding. Molecular weight (MW) markers are indicated at left. Results are representative of at least three biological replicates (see Supplementary Fig. 4 for replicate data)
