High-level production of prourokinase-annexin V chimeras in the methylotrophic yeast Pichia pastoris

Prourokinase (proUK)-annexin V chimeras expressed by the methylotrophic yeast Pichia pastoris in a synthetic medium as part of a system designed to yield a novel thrombolytic agent are degraded, as it is thought, by various yeast proteases present in the culture supernatant. Minimization of proteolysis was therefore investigated to increase the yield of intact proUK-annexin V. Protease inhibitor screening study indicated several proteases including at least serine protease like chymotrypsin were involved in the proteolysis. Addition of more than 10% of peptone or more than 0.2 mol l(-1) of arginine to the medium was effective in minimizing proteolysis in shake-flask culture. Culture condition of higher pH was also effective, however, induced a cell death. Cell improvement by increasing the methanol utilization ability yielded greater tolerance to high pH. As a result, the culture condition with highly concentrated peptone solution fed under controlled conditions of pH 8.0 was established, which greatly reduced proteolytic degradation in fed-batch fermentation. These optimal conditions, which enabled fibrinolytic activity to reach 7800 IU ml(-1), could easily be applied in industrial scale production.