EXPRESSION OF THE HUMAN UROKINASE-TYPE PLASMINOGEN-ACTIVATOR RECEPTORIN ESCHERICHIA-COLI AND CHINESE-HAMSTER OVARY CELLS - PURIFICATION OFTHE RECOMBINANT PROTEINS AND GENERATION OF POLYCLONAL ANTIBODIES IN CHICKEN

The receptor for urokinase-type plasminogen activator (uPAR) may contr ibute to the invasive and metastatic capacity of tumor cells by focusi ng the serine protease urokinase-type plasminogen activator (uPA) to t he cell surface. uPA activates plasminogen to plasmin which in turn de grades extracellular matrix proteins or activates other proteases. Mat ure uPAR is a heavily glycosylated protein of about 284 amino acids at tached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI ) anchor. A set of different polyclonal uPAR antibodies has been gener ated in order to investigate the role of uPAR in tumor spreading in mo re detail. For this purpose, uPAR (lacking the GPI anchor) was express ed in E. coli and Chinese hamster ovary (CHO) cells. Recombinant uPAR from E. coli (corresponding to amino acids 1-284 of human uPAR) was ex pressed with an N-terminal histidine-tag insertion and purified by nic kel chelate affinity chromatography. Soluble uPAR, synthesized by CHO cells (corresponding to amino acids 1-277 of human uPAR), was isolated by ligand (uPA) affinity chromatography. Expression in E. coli led to a nonglycosylated form of uPAR, whereas uPAR produced by CHO cells se emed to be glycosylated to a similar extent as the naturally occurring human form of uPAR (as analyzed by sodium dodecyl sulfate-polyacrylam ide gel electrophoresis). Prior to immunization the N-termini of the r ecombinant uPAR variants were determined by amino acid sequence analys is. Polyclonal antibodies were generated in chickens and purified from egg yolk. The reaction patterns of these antibodies were analyzed by Western blot analyses and flow cytofluorometry.