Quantitative Protein Analysis from Cell Cultures

The total protein content from culture media and cell fraction was assessed as described previously (Krasauskas et al., 2019 (link)) with some modifications. Briefly, strains were grown stationary in TSB media at 30°C for 30 h. The cells were separated from the media by a centrifugation at 10,000 × g for 10 min at 4°C. The supernatant was further clarified by a filtration through 0.22 μm filter to remove the remaining biomass. The proteins were precipitated by the addition of trichloroacetic acid (TCA) to a final concentration of 10% and centrifuged at 15,000 × g for 60 min at 4°C. The pellet was washed twice with an ice-cold acetone and dried by incubating tube at 95°C. The separated cells and precipitated proteins were then re-suspended with Laemmli-12% SDS-PAGE sample buffer. After electrophoresis, gels were stained with Coomassie brilliant blue. The PageRuler^TM unstained broad range protein ladder (5 μl per lane) was used as a marker (Thermo Fisher Scientific).