Pre-Golgi degradation of yeast prepro-alpha-factor expressed in a mammalian cell. Influence of cell type-specific oligosaccharide processing on intracellular fate.

We demonstrate that expression of yeast prepro-alpha-factor in GH3 rat pituitary cells results in its degradation in an endoplasmic reticulum (ER) or early Golgi compartment, suggesting that this wild-type prohormone is recognized as abnormal or misfolded in the context of a mammalian ER. In GH3 cells, as in yeast, the nascent polypeptide is efficiently targeted to the ER, where it undergoes cleavage of its amino-terminal signal peptide and core glycosylation to form glycosylated pro-alpha-factor (gp alpha f). Subsequently, however, this species disappears from cells with a half-time of 25-30 min (including a 10-20-min lag), and no alpha-factor- or proregion-derived products can be detected.

Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.

Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2).