Extended Data Fig. 2: Workflows for probe production, determination of O-glycopeptidase activity, and assaying for GH activity.
From: A high-throughput screening platform for enzymes active on mucin-type O-glycoproteins
a, Origami2(DE3) cells (or the OG2neu+ strain) are transformed with the probe plasmid and then the OGO of choice. Protein expression is carried out by IPTG induction. The probe can then be obtained in sufficient purity via Ni-NTA chromatography, with subsequent completion of chromophore maturation being carried out by incubation at 37 °C as needed. b, Kinetic characterization of O-glycopeptidases using purified probe. Briefly, v0 values are determined for a set of different substrate concentrations. A corresponding standard curve with the expected changes in FRET then enables unit conversion to M/s or mg/mL/s. c, Screening for GH activity in plates using MELiORA. E. coli carrying a plasmid encoding the GH of interest are grown and induced in supplemented M9 media with IPTG. They are then lysed in the presence of the probe and incubated to allow for the GHs to act on the probe. Following this, the glycosylation status of the probe is determined by addition of an O-glycopeptidase with subsequent read-out of the FRET in a plate reader.
