FIG. 6.
Immunotoxin processing and secretion. Immunotoxin was expressed under PAOX1 control in P. pastoris strains containing one copy (strain PAOX-1c) (A) and two copies (strain PAOX-2c) (B) of the gene. Cells were labeled for 7 min, chased for the times indicated, and then lysed. The cell lysates and culture media were immunoprecipitated with anti-DT antibody before SDS-PAGE and phosphorimaging. The primary translation product of the immunotoxin gene contains α-factor pre- and prosequences. The presequence is removed when the polypeptide enters the ER to generate proimmunotoxin (Pro-IT), which is then further processed to yield mature immunotoxin (IT). Two major immunotoxin breakdown products were identified: immunotoxin without the diphtheria toxin A chain (IT-DTA) and the DT A chain (DTA). The intensities of the Pro-IT and IT bands in panels A and B were measured by using ImageQuant software, and the results are shown in panel C.