Strategy for screening the N-TIMP2RGD library. (A) YSD system. N-TIMP2RGD proteins were displayed on yeast. Expression of the N-TIMP2RGD proteins was detected by targeting the C-Myc epitope tag, expressed at the C-terminal of the protein, with an antibody labeled with phycoerythrin (PE), and binding to integrin αvβ3 was detected by FITC staining. (B) Flow cytometry analysis of the sorted library tested against 50 nM of integrin αvβ3. In the expression sort (ES), the entire N-TIMP2RGD protein-expressing cell population was sorted (black gate). In sort 1 (S1), the yeast cell population binding to integrin αvβ3 was collected (black gate). In sort 2 (S2) to sort 5 (S5), the integrin αvβ3 high affinity population was collected (black gate). The population binding integrin αvβ3 was enriched as the sorting progressed from S2 to S5. (C) N-TIMP2 protein variants. N-TIMP2WT,RGD is the bi-specific protein isolated after sort 5. N-TIMP25M,RGD is N-TIMP2WT,RGD with 5 mutations (5M); this mutant showed improved binding to MMP-14 (39). N-TIMP2HD is a heterodimer composed of N-TIMP25M and N-TIMP2WT,RGD proteins conjugated via a peptide linker. N-TIMP2WT and N-TIMP25M are mono-specific binders for MMP-14. Ala-N-TIMP2WT,RGD has a reduced affinity toward MMP-14 and high affinity toward integrin avb3.