title: Expression and in vitro activation of Manduca sexta prophenoloxidase-activating proteinase-2 precursor (proPAP-2) from baculovirus-infected insect cells.
abstract: Prophenoloxidase activation is a component of the immune system in insects and crustaceans. We recently purified and cloned a new prophenoloxidase-activating proteinase (PAP-2) from hemolymph of the tobacco hornworm Manduca sexta [J. Biol. Chem. 278, 3552-3561]. As the terminal component of a putative serine proteinase cascade, this enzyme activates prophenoloxidase (proPO) via limited proteolysis. To purify and study the activating proteinase for PAP-2 from this insect, we expressed the zymogen of PAP-2 (proPAP-2) in insect cells infected by a recombinant baculovirus that harbors the cDNA. To facilitate the purification of proPAP-2, we modified a commercial vector (pFastBac1) by inserting a synthetic DNA fragment encoding a hexahistidine sequence, allowing fusion of the affinity tag to the carboxyl terminus of a protein. After Spodoptera frugiperda Sf21 cells were infected by the virus, recombinant proPAP-2 was efficiently secreted into the media at a concentration of 5.9 microg/ml under the optimal conditions. After ammonium sulfate precipitation, the proenzyme was purified to near homogeneity by affinity chromatography on Ni(2+)-NTA agarose. Western blot analysis indicated that the recombinant proPAP-2 has a mobility slightly lower than that of the zymogen from M. sexta hemolymph. The molecular mass and isoelectric point of proPAP-2 were determined to be 47,573+/-11Da and 6.6, respectively. After the purified proenzyme was added to hemolymph from induced M. sexta larvae, it was rapidly activated by an unknown proteinase in the presence of peptidoglycan.
authors.affiliation: Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Chuanyi
authors.initials: C
authors.lastname: Ji
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Jeremy
authors.initials: J
authors.lastname: Ross
authors.forename: Haobo
authors.initials: H
authors.lastname: Jiang
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004156:Catechol Oxidase / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D018402:Manduca / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D018411:Spodoptera / Q000821:virology
chemical_list: D004792:Enzyme Precursors; D011994:Recombinant Proteins; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D012697:Serine Endopeptidases; D000581:Amidohydrolases; C019665:amidase