Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.
Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.
Kost
T A
TA
Molecular Sciences Department, Glaxo Wellcome Research Institute, Research Triangle Park, NC 27709, USA. tak4306@glaxo.com
Ignar
D M
DM
Clay
W C
WC
Andrews
J
J
Leray
J D
JD
Overton
L
L
Hoffman
C R
CR
Kilpatrick
K E
KE
Ellis
B
B
Emerson
D L
DL
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D010960:Plasminogen Activators / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D018411:Spodoptera; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator