High level synthesis and secretion of human urokinase using a late gene promoter of the Autographa californica nuclear polyhedrosis virus.
A cDNA encoding human urokinase was inserted into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome at the polyhedrin gene locus under control of a duplicated copy of the late, basic protein gene promoter. The insect-derived urokinase was produced predominantly in the form of single-chain, pro-urokinase, with a molecular mass of 50 kDa, and demonstrated fibrinolytic activity. Synthesis and secretion of urokinase was first detected at 6 hours post-infection and continued steadily throughout the infection period. Comparisons with urokinase synthesised using the very late AcNPV polyhedrin gene promoter revealed that, although the polyhedrin promoter is intrinsically stronger, the yield of secreted urokinase was higher using the basic protein gene promoter. These data support the hypothesis that the host cell secretory pathway is compromised in the very late stages of baculovirus infection and may provide an explanation for why, in general, secreted and membrane-targeted proteins are not produced to the high levels observed with other proteins, when using very late baculovirus gene promoters.
Lawrie
A M
AM
School of Biological and Molecular Sciences, Oxford Brookes University, UK.
King
L A
LA
Ogden
J E
JE
D000818:Animals; D001709:Biotechnology; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D000077183:Occlusion Body Matrix Proteins; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D018411:Spodoptera; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D018076:DNA, Complementary; D000077183:Occlusion Body Matrix Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D014568:Urokinase-Type Plasminogen Activator