One-step affinity purification of recombinant urokinase-type plasminogen activator receptor using a synthetic peptide developed by combinatorial chemistry.
Several lines of evidence have pointed to a role of urokinase-type plasminogen activator receptor (uPAR) as a modulator of certain biochemical processes that are active during tumor invasion and metastasis. Consequently, the structure and function of this receptor have been studied extensively, using recombinantly produced uPAR that has been purified by either affinity chromatography using its cognate ligand, the urokinase-type plasminogen activator (uPA), or a monoclonal anti-uPAR antibody (R2), or by hydroxyapatite. Here, we present a new method for the efficient one-step affinity purification of recombinant uPAR exploiting a high-affinity synthetic peptide antagonist (AE152). The corresponding parent peptide was originally identified in a random phage-display library and subsequently subjected to affinity maturation by combinatorial chemistry. This study compares the affinity purification of a soluble, recombinant uPAR using the monoclonal antibody R2 or the peptide AE152 immobilized on Sepharose. The two affinity ligands perform equally well in purifying uPAR from Drosophila melanogaster Schneider 2 cell culture medium and yield products of comparable purity, activity, and stability as judged by SDS-PAGE, size exclusion chromatography and surface plasmon resonance analysis. The general availability of peptide synthesis renders the present AE152-based affinity purification of uPAR more accessible than the traditional protein-based affinity purification strategies. In this way, large amounts of recombinant uPAR can conveniently be purified for further structural and functional studies.
Jacobsen
Benedikte
B
Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark.
Gårdsvoll
Henrik
H
Juhl Funch
Gitte
G
Ostergaard
Søren
S
Barkholt
Vibeke
V
Ploug
Michael
M
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D020349:Surface Plasmon Resonance; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D008024:Ligands; D010455:Peptides; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator