Molecular design and biochemical characterization of the scFv/uPA-T fusion protein. (A) Single-chain variable fragment (scFv) fused with thrombin-inducible lmw-scuPA (scFv/uPA-T) was generated by deleting Phe¹⁵⁷ and Lys¹⁵⁸ from the previously described construct, scFv/uPA. This converts the plasmin activation site Pro¹⁵⁵-Arg¹⁵⁶-Phe¹⁵⁷-Lys¹⁵⁸-Ile¹⁵⁹-Ile¹⁶⁰ (PRFKII) into the sequence Pro¹⁵⁵-Arg¹⁵⁶-Ile¹⁵⁷-Ile¹⁵⁸ (PRII), which is cleaved by thrombin after Arg¹⁵⁶. (B) Migration of the purified fusion protein in the absence or presence of thrombin was analyzed using SDS-PAGE under nonreduced or reduced conditions.