PRODUCTION OF A UROKINASE PLASMINOGEN ACTIVATOR-IGG FUSION PROTEIN (UPA-IGG) IN THE BACULOVIRUS EXPRESSION SYSTEM

Numerous studies have demonstrated the importance of urokinase plasmin ogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may repres ent an important target for inhibiting metastatic disease. The baculov irus expression system was used to produce high levels of a secreted u PA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uP A-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter, Rec ombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uP A-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 mu g/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h postinfection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 mu g /ml. The amount of cell-associated uPA-IgG in infected Bn-Tn-5B1-4 cel ls was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-I gG was readily purified using a combination of zinc chelate and sephac ryl S-100 column chromatography. Routinely, greater than 100 mg of gre ater than 95% pure protein could be obtained per liter of culture medi um collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the bas ic protein promoter virus. BIAcore analysis and competition binding as says using LOX human malignant melanoma cells expressing uPAR indicate d that the purified recombinant protein possessed similar ligand bindi ng characteristics to that of human uPA.