Expression and purification of Manduca sexta prophenoloxidase-activating proteinase precursor (proPAP) from baculovirus-infected insect cells

Analogous to human thrombin, prophenoloxidase-activating proteinase (PA-P) is a terminal enzyme of a serine proteinase cascade in the tobacco hornworm Manduca sexta. In order to purify and study the activating enzyme for PAP from this insect, we produced the zymogen of PAP (proPAP) in a bacterial ex pression system. The affinity-purified protein was then used as an antigen to generate a specific rabbit antiserum. Immunoblot analysis indicated that the proPAP was present at a low level in Manduca larval hemolymph, but was induced by six- to eightfold in larvae that had been injected with Escheri chia coli or Micrococcus lysodeikticus. To produce the native proenzyme for functional analyses, we constructed a recombinant baculovirus to infect Sp odoptera frugiperda Sf21 cells. ProPAP was secreted into the medium at a lo w concentration of approximately 0.37 mg/liter under the optimal conditions . We then developed a simple, efficient scheme to enrich and purify this pr otein, which involves two lectin affinity and one HPLC ion-exchange chromat ographic steps. Immunoblot analysis following SDS-polyacrylamide gel electr ophoresis indicated that the recombinant proPAP is nearly identical in mobi lity to the zymogen from Manduca hemolymph. After the purified proPAP was a dded to the larval hemolymph, it was readily activated by an unknown protei nase in the presence of M. lysodeikticus. (C) 2001 Academic Press.