Chymosin, an aspartyl protease, is secreted as a zymogen, prochymosin, which is autocatalytically activated to chymosin at low pH. The enzyme is naturally found in the fourth stomach of the calf and cleaves kappa-casein. We took advantage of this milk clotting activity to rapidly screen transformants for expression and secretion of chymosin. Figure 1 . Steps in the construction of the chymosin expression plasmids are described in the text. Presented are the expression cassettes for each plasmid.
Open arrow represents preprochymosin sequences.
Histidine prototrophs were selected on minimal medium, and transformants were transferred to agar slants. Figure 2 . Western transfer of medium from various N. crassa transformants. A. Lane 1. Molecular weight markers.