Figure 3
From: Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli
Determination of the flavinylated vs. non-covalent recombinant mBCO by means of SDS-PAGE analysis. SDS-PAGE Gel was analyzed for flavin fluorescence (panel A) and subsequently stained for total proteins with Coomassie-blue (panel B). Conditions: recombinant E. coli cells grown in LB medium, added of 1 mM IPTG at OD600 nm = 0.9, then incubated at 25°C and collected after 18 hours. I, Insoluble fraction (pellet after cell disruption and centrifugation); S, soluble fraction (soluble crude extract). An amount of sample corresponding to 0.1 mL of fermentation broth was loaded in each lane. BCOcov and BCOncov: purified flavinylated and non-covalent mBCO forms, respectively; the amount of enzyme loaded are indicate in micrograms. M: molecular weight markers (indicated in kDa on the right side of panel B).