Figure 4

UPR response in secretory insulin precursor producing P. pastoris X-33-IP and non-producing host strains X-33 and GS115. Cells were grown on glycerol in shake flask cultures (same glycerol concentration as in bioreactor cultures) and resuspended in medium containing methanol. Samples were taken at the end of the glycerol phase directly before induction with methanol (0) and at 72 and 96 h after induction with 1% or 2% methanol. (A) Crude cell lysates from 1% methanol cultures of the secretory insulin precursor producing P. pastoris X-33 (X-33-IP), and the non-producing host strains P. pastoris X-33 (X-33-host) and GS115 (GS115-host) were analyzed by SDS-PAGE. (B) Crude cell lysates from 1% and 2% methanol cultures of the secretory insulin precursor producing P. pastoris X-33 (X-33-IP) and the non-producing host P. pastoris X-33 (X-33-host) were probed for proteins containing the endoplasmic reticulum retention signal peptide HDEL (e.g. KAR2, 74 kDa and PDI, 58 kDa) by Western Blot analysis.