Molecular design, expression, and characterization of anti-PECAM scFv-lmw scuPA. (A) Schematic diagram describing the cloning strategy for the fusion construct pMT-BD1. Variable domains of heavy chain and light chain were linked by a (Gly₄Ser)₃ linker and then fused to the N-terminus of lmw-scuPA by a (Ser₄Gly)₂Ala₃ linker. (B) Variable domains of heavy chain (H chain) and light chain (L chain) of P-390 were amplified and assembled into full-length scFv. M indicates DNA standards. (C) Lmw-scuPA and anti-PECAM scFv were ligated and cloned into SpeI and XhoI sites of the pMT expression vector. XhoI and SpeI digestion of the fusion construct (fusion). (D) Western blot analysis of 40 μL culture medium alone or after induction by 0.5 mM CuSO₄. Purified fusion protein (50 ng and 200 ng) was blotted to compare expression levels. (E) Four percent to 12% gradient SDS-PAGE of purified fusion protein with or without plasmin treatment under unreduced or reduced conditions.