FIG. 1.

Native secreted subtilisin produced by P. pastoris GS115, KM71, and derivative strains in methanol medium. (Left panel) Ten microliters of P. pastoris culture supernatant was loaded onto the gel (lane 1, GS115-sub2; lane 2, KM71-sub2; lane 3, GS115; lane 4, KM71). (Right panel) P. pastoris GS115 and KM71 were grown to near saturation (OD of 20 at 600 nm) at 30°C in 10 ml of glycerol-based yeast medium for 2 days. Cells were harvested by centrifugation and resuspended in 2 ml of the same medium with 0.5% (vol/vol) methanol instead of glycerol and incubated for 2 days. For lanes 5 (GS115) and 6 (KM71), 10 μl of 50×-concentrated culture supernatant in glycerol-based yeast medium was loaded onto the gel. For lanes 7 (GS115) and 8 (KM71), 10 μl of 10×-concentrated culture supernatant in methanol medium as appropriate controls was loaded onto the gel. MM, molecular mass.