Identifying substrates for endothelium-specific Tie-2 receptor tyrosine kinase from phage-displayed peptide libraries for high throughput screening

The peptide substrate specificity of Tie-2 was probed using the phage displ ay method in order to identify efficient substrate for high throughput scre ening. Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructe d, in which all twenty amino acid residues were represented at the X positi ons flanking the fixed tyrosine residue Y. A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylati on. The phosphorylated phage particles were enriched by panning over immobi lized antiphosphotyrosine antibody pY20 for a total of 5 rounds. Four phage clones (3T61, 3T68, C-90 and D1-15) that express a peptide sequence that c an be phosphorylated by the recombinant catalytic domain of human Tie-2 wer e identified. Synthetic peptides made according to the sequences of the 4 s elected clones from the two libraries, which had widely different sequences , were active substrates of Tie-2. Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a k(cat)/K-m of 5.9x10(4) M-1 s(-1). Th ree high throughput screening assay formats, dissociation-enhanced lanthani de fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-res olved fluorescent resonance energy transfer (TR-FRET) were developed to ass ess the suitability of these phage display selected peptides in screening T ie-2 inhibitors. Three out of four peptides were functional in the DELFIA a ssay and D1-15 was functional in the TR-FRET assay.