Expression of human plasminogen cDNA in a baculovirus vector-infected insect cell system.
A cDNA that encodes the human plasminogen (HPg) amino acid sequence has been inserted adjacent to the polyhedrin promoter in the genome of the baculovirus, Autographa californica nuclear polyhedrosis virus, which was then used to infect cultured cells of the farm armyworm, Spodoptera frugiperda. Under the conditions of cell growth employed, recombinant (rec)-HPg was secreted into the medium after 24 h postinfection (p.i.), at which point virtually no rec-HPg antigen remained inside the cells. At 48 h p.i., a maximal level of intact rec-HPg was present in the medium, which underwent substantial proteolytic digestion after that time. The rec-HPg produced by this expression system possessed a molecular weight equivalent to that of plasma [Glu1]-plasminogen. In addition, the rec-HPg adsorbed to Sepharose-lysine, and was eluted with epsilon-aminocaproic acid (EACA). The recombinant protein also interacted with polyclonal antibodies generated to plasma HPg, as well as with a monoclonal antibody directed against a distinct region (kringle 1-3) of the plasma HPg molecule. Finally, the insect-expressed rec-HPg was activatable to plasmin (HPm) by urokinase. The results demonstrate that this expression system produces a full-length functional single-chain rec-HPg, which can be isolated intact from the culture medium, with some consideration for the temporal events that occur in secretion and longer-term degradation of the protein. The fact that this rec-HPg was converted to HPm with a plasminogen activator, and that it interacted with anti-plasma HPg polyclonal and monoclonal antibodies, as well as with the ligand, EACA, indicates that the molecule retains many of its important functional properties and is folded in an integral manner.
Whitefleet-Smith
J
J
Department of Chemistry, University of Notre Dame, Indiana 46556.
Rosen
E
E
McLinden
J
J
Ploplis
V A
VA
Fraser
M J
MJ
Tomlinson
J E
JE
McLean
J W
JW
Castellino
F J
FJ
D000818:Animals; D000845:Annelida / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007304:Insect Viruses / Q000235:genetics*; D007700:Kinetics; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010958:Plasminogen / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology
D011994:Recombinant Proteins; D010958:Plasminogen; D004247:DNA; D014568:Urokinase-Type Plasminogen Activator