FIG. 3.
Relative secretion levels for immunotoxin and Kar2p. Protein expression was performed as described in Materials and Methods for 22 h. (A) Coomassie blue-stained SDS gel of culture supernatants of P. pastoris strains expressing genes for cytosolic β-galactosidase (lane β-gal) or secreted serum albumin (lane SA) and for immunotoxin (lanes 1c, one copy; lanes 2c, two copies) under the control of PAOX1 or PGAP. Lanes X-33 contained wild-type strain X-33 culture supernatants. IT, immunotoxin. (B) Western blots of culture supernatant of the strain expressing one copy of the immunotoxin gene (PGAP-1c). The blots were probed with the anti-Kar2p antibody (left blot) or anti-G4S antibody (right blot). Unlike anti-DT antibody, anti-G4S reacts equally well with intact immunotoxin and immunotoxin devoid of the diphtheria toxin A chain (IT-DTA).