HIGH-LEVEL SECRETION OF NATIVE RAT PANCREATIC LITHOSTATHINE IN A BACULOVIRUS EXPRESSION SYSTEM

We have constructed a recombinant baculovirus expression vector contai ning rat pancreatic lithestatin cDNA. Baculovirus infection of Spodopt era frugiperda (sf9) insect cells resulted in the de novo synthesis an d secretion of a recombinant protein demonstrating an apparent molecul ar weight of about 16.5 kDa. Under optimal conditions [multiplicity of infection of 5 plaque-forming units (pfu)/cell and culture times of 4 8-56 h postinfection] recombinant protein was secreted into the cultur e medium at 5-10 mg/L. The secretory form of the recombinant protein w as judged to be rat pancreatic lithostatin by the following criteria: (a) Trypsin cleavage resulted in limited proteolysis of the secreted p roduct giving rise to a trypsin-resistant 15.5-kDa peptide, consistent with the size of the ''pancreatic stone/thread protein''; (b) polyclo nal antibodies raised against the recombinant protein identified 16.5- kDa secretory proteins in both rat pancreatic juice and sf9 culture me dium; and (c) immunohistochemistry indicated that the native antigen r esides within zymogen granules in pancreatic acinar cells.