Design and characterization of 1-11E/vIL-10 fusion. (A) Schematic representation of the 1-11E/vIL10 fusion protein, as cloned into the pcDNA expression vectors. SP, signal peptide. The scFv and vIL-10 are separated by an MMP cleavage linker, whereas the His-tag and V5-tag were incorporated at the C-terminus. (B) SDS-PAGE analysis of purified 1-11E/vIL-10 is shown in the left panel. Purified 1-11E/vIL-10 fusion protein migrated at approximately 50 kDa, in keeping with the expected molecular weight (~30 kDa scFv plus ~20 kDa vIL-10). Digestion of 1-11E/IL10 by MMPs is shown in the right panel. 1-11E/vIL-10 fusion protein was incubated with the catalytic domains of MMP-1, MMP-3, and MMP-12 overnight and analyzed with Western blot by using anti-tetra-His-HRP antibodies. (C) FPLC profile of 1-11E/IL10. The first major peak at approximately 100 kDa corresponds to the dimer form, and the second minor peak at approximately 50 kDa corresponds to the monomer form.