YEAST EXPRESSION AND PHAGEMID DISPLAY OF THE HUMAN UROKINASE PLASMINOGEN-ACTIVATOR EPIDERMAL GROWTH FACTOR-LIKE DOMAIN

The human urokinase plasminogen activator (uPA) epidermal growth facto r-like domain (residues 1-48) and a variant with a C-terminal epitope tag have been secreted from recombinant yeast, Purified human uPA 1-48 and uPA 1-48glu compete for binding to the human uPA receptor with K( d)s of 180 and 400 pM respectively, in an in vitro assay using an immo bilized recombinant uPA receptor. A synthetic gene encoding human uPA 1-48 with an N-terminal epitope tag was inserted into a phagemid expre ssion vector as a fusion with residues 249-406 of the M13 pIII protein with an intervening amber codon (TAG). Phagemid production led to inf ectious particles which were selectively bound and eluted from both ep itope tag antibody and urokinase receptor. Sequential binding to this antibody and receptor demonstrated a substantial enrichment, where up to 10% of the infectious particles were then retained on urokinase rec eptor-coated plates, A PCR strategy was used to convert previously des cribed peptide bacteriophage ligands for the urokinase receptor to pha gemid display. The yields of these peptide phagemids and the uPA 1-48 phagemid showed a correlation with peptide affinity, in contrast to wh en the peptides are multivalently displayed on a bacteriophage.