Figure 4. Cell-free O-glycosylation using glyco-enriched extracts.

(a) Immunoblot analysis of in vitro glycosylation (IVG) reactions that were performed by incubating purified MBP^MOOR or MBP^MOORmut acceptor proteins in the presence of crude membrane extracts (CMEs) prepared from CLM25 cells carrying pOG-T-NgPglO (+) or pOG-T without an O-OST (−). Glyco-enriched CMEs alone (lane 1) or glycosylated MBP^MOOR (gMBP^MOOR) that was previously purified from glycoengineered bacteria (lane 5) served as negative and positive controls, respectively. (b) Immunoblot analysis of acceptor proteins produced by integrated CFGpS in which transcription, translation, and O-glycosylation were performed altogether in a single reaction. Specifically, 1 ml reactions comprised of glyco-enriched S12 extract derived from CLM25 cells carrying pOG-T-NgPglO were primed with plasmid pJL1-MBP^MOOR or pJL1-MBP^MOORmut as indicated. Blots in (a) and (b) were probed with anti-hexa-histidine antibody (6xHis) to detect the acceptor proteins and PNA to detect the T antigen. Molecular weight (MW) markers are indicated on the left. Results are representative of at least three biological replicates. See Supplementary Fig. 2 for uncropped versions of the images.