Purification and characterization of recombinant human pro-urokinase produced in silkworm using a baculovirus vector

Summary A recombinant baculovirus (BmNPV-pk2) was constructed by inserting the human pro-urokinase(pro-UK) cDNA into the genome of baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) adjacent to the strong polyhedrin promoter. The recombinant virus replicated in silkworm larvae, which synthesized 30g pro-UK/ml in the haemolymph within 4 days post-infection. Purification to near homogeneity was accomplished by fractional precipitation with ammonium sulphate and immunoaffinity chromatography with an overall yield of 23% and a specific activity of 100,000IU/mg in fibrin plate assay. This purified product was comprised of a single chain protein with approximately Mr. 50kDa as determined by SDS-PAGE gel. The N-Terminal amino acids sequence revealed that the secretion signal of pro-UK was correctly processed.