Fig. 4

The protein level of Mut M is higher both in cellulo and in culture medium. a SDS-PAGE analysis of total intracellular proteins extracted from P. pastoris wild-type SMD 1168 (SMD), WT D2 and Mut M strains. Ten micrograms of proteins were loaded for each sample. b Western blotting analysis of total intracellular proteins with specific antibodies anti-D2-BGL. c Western blotting of culture supernatant collected from WT D2 and Mut M at 24-h interval. Twenty microliters of supernatant were loaded in each well. d Western blotting with deglycosylated culture supernatant from WT D2 and Mut M. Culture supernatants from three different clones of Mut M (Mut M -1, -2 and -3) and WT D2 (WT D2 -1, -2 and -3) were used. Four microliters of deglycosylated sample were loaded in each well. Samples were collected from a 3-day culture. Protein concentration was estimated using purified D2-BGL as standard