Yeast expression and phagemid display of the human urokinase plasminogen activator epidermal growth factor-like domain.
The human urokinase plasminogen activator (uPA) epidermal growth factor-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 complete for binding to the human uPA receptor with Kds of 180 and 400 pM respectively, in an in vitro assay using an immobilized recombinant uPA receptor. A synthetic gene encoding human uPA 1-48 with an N-terminal epitope tag was inserted into a phagemid expression vector as a fusion with residues 249-406 of the M13 pIII protein with an intervening amber codon (TAG). Phagemid production led to infectious particles which were selectively bound and eluted from both epitope 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 receptor-coated plates. A PCR strategy was used to convert previously described peptide bacteriophage ligands for the urokinase receptor to phagemid display. The yields of these peptide phagemids and the uPA 1-48 phagemid showed a correlation with peptide affinity, in contrast to when the peptides are multivalently displayed on a bacteriophage.
Chiron Corporation, Emeryville, CA 94608, USA.
J R
JR
Stratton-Thomas
H Y
HY
Min
S E
SE
Kaufman
C Y
CY
Chiu
G T
GT
Mullenbach
S
S
Rosenberg
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D005813:Genes, Synthetic; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D000939:Epitopes; D008024:Ligands; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D014568:Urokinase-Type Plasminogen Activator