High-level secretion of native rat pancreatic lithostathine in a baculovirus expression system.
We have constructed a recombinant baculovirus expression vector containing rat pancreatic lithostatin cDNA. Baculovirus infection of Spodoptera frugiperda (sf9) insect cells resulted in the de novo synthesis and secretion of a recombinant protein demonstrating an apparent molecular weight of about 16.5 kDa. Under optimal conditions [multiplicity of infection of 5 plaque-forming units (pfu)/cell and culture times of 48-56 h postinfection] recombinant protein was secreted into the culture medium at 5-10 mg/L. The secretory form of the recombinant protein was judged to be rat pancreatic lithostatin by the following criteria: (a) Trypsin cleavage resulted in limited proteolysis of the secreted product giving rise to a trypsin-resistant 15.5-kDa peptide, consistent with the size of the "pancreatic stone/thread protein"; (b) polyclonal antibodies raised against the recombinant protein identified 16.5-kDa secretory proteins in both rat pancreatic juice and sf9 culture medium; and (c) immunohistochemistry indicated that the native antigen resides within zymogen granules in pancreatic acinar cells.
Bimmler
D
D
Laboratory of Cell and Molecular Biology, Charles A. Dana Research Institute, Boston, Massachusetts, U.S.A.
Frick
T W
TW
Scheele
G A
GA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D005822:Genetic Vectors; D007150:Immunohistochemistry; D051847:Lithostathine; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D010179:Pancreas / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera; D014357:Trypsin / Q000378:metabolism
D002135:Calcium-Binding Proteins; D051847:Lithostathine; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C497616:Reg1a protein, rat; D014357:Trypsin