Schematic diagrams of construction of phage display library and biopanning strategy.
Panel A shows the cloning strategy employed to create T7Select10-3b phage constructs displaying API M358R or libraries displaying API with randomized RCL codons. 1) The API M358R cDNA was mobilized from pBAD-H6API M358R  by PCR that appropriately positioned EcoRI (green) and HindIII cohesive ends, and ensured that it was in-frame for subsequent insertion into the T7 Select 10-3b vector, in plasmid pUC19-API M358R. 2) Degeneracy was introduced at either P2–P1 or P7–P3 codons using a sense primer overlapping the unique PmlI site or a degenerate antisense (NNN primer) overlapping the unique SauI site. PCR products were inserted into phosphatase-treated PmlI- and SauI-restricted pUC19-API M358R and the resulting plasmid library biologically amplified in E. coli TOP10. 3) EcoRI-and HindIII-restricted total digestion products of the P2–P1 or P7–P3 plasmid libraries, respectively, were then ligated to T7Select10-3b vector arms. 4) The recombinant T7Select10-3b library, containing either the API P2–P1 randomized or the API M358R P7–P3 randomized inserts, was then packaged into phages to create the respective phage display libraries. Pink, light blue, yellow, and purple API-encoding inserts (between steps 2 and 3) indicate the encoding of different variants, and the correspondingly coloured phages (step 4) represent their displayed products. Panel B shows the biopanning procedure. 1) A phage lysate produced by infection of E. coli BLT5615 with a T7Select10-3b API library was reacted with thrombin in solution (green squares), which bound to some displayed sequences (e.g. blue but not yellow or pink). 2) A biotinylated antibody specific to thrombin (Y-shaped symbols) was added to the thrombin-phages mixture, reacting with thrombin bound to phages via API-thrombin serpin-enzyme complexes. 3) Streptavidin-coated magnetic beads (yellow S shown on red circular symbols) were added to the mixture, reacting preferentially with antibody-thrombin-phage complexes. 4) Magnetic bead-streptavidin-thrombin-phage complexes were concentrated using a magnet. 5) After washing, bead assemblies were used to directly infect a fresh culture of E. coli BLT5615 to start the next round of biopanning.