Figure 1.

Strategy for screening the N-TIMP2_RGD library. A, YSD system. N-TIMP2_RGD proteins were displayed on yeast. Expression of the N-TIMP2_RGD proteins was detected by targeting the c-Myc epitope tag, expressed at the C terminus of the protein, with an antibody labeled with PE, and binding to integrin α_vβ₃ was detected by FITC staining. B, flow cytometry analysis of the sorted library tested against 50 nm integrin α_vβ₃. In the expression sort (ES), the entire N-TIMP2_RGD protein–expressing cell population was sorted (black gate). In sort 1 (S1), the yeast cell population binding to integrin α_vβ₃ was collected (black gate). In sort 2 (S2) to sort 5 (S5), the integrin α_vβ₃ high-affinity population was collected (black gate). The population binding integrin α_vβ₃ was enriched as the sorting progressed from S2 to S5. C, N-TIMP2 protein variants. N-TIMP2_{WT, RGD} is the bi-specific protein isolated after sort 5. N-TIMP2_{5M, RGD} is N-TIMP2_{WT, RGD} with five mutations (5M); this mutant showed improved binding to MMP-14 (38). N-TIMP2_HD is a heterodimer composed of N-TIMP2_5M and N-TIMP2_{WT, RGD} proteins conjugated via a peptide linker. N-TIMP2_WT and N-TIMP2_5M are mono-specific binders for MMP-14. Ala–N-TIMP2_{WT, RGD} has a reduced affinity toward MMP-14 and a high affinity toward integrin α_vβ₃.