FIG. 4.
Secretion of the immunotoxin increases Kar2p secretion. Protein expression was performed for 4 h under conditions described in Materials and Methods. P. pastoris strains expressing immunotoxin (lanes 1c, one copy; lanes 2c, two copies) under PGAP control (A and B) were incubated in YPD and buffered (pH 7.0) richer expression medium with dextrose (B-EMD), whereas strains expressing immunotoxin, serum albumin (lane SA), or β-galactosidase (lane β-gal) under PAOX1 control (C) were induced in expression medium containing methanol (B-EMM). Lanes X-33 contained wild-type strain X-33. Samples of culture supernatant were analyzed by SDS-PAGE and Western blotting using anti-Kar2p (A and C) or anti-DT (B) as the primary antibody.