Expression and identification of fused proteins. A: Construction of recombinant expression plasmids. pET32a(+)/MIL5scFv and pET32a(+)/MIL5scFv-Arg9. B: Gene amplification of scFvs by PCR. Lane 1: MIL5scFv; lane 2: MIL5scFv-Arg9; lane 3: DNA marker DL2000 plus. C: SDS-PAGE analysis of purified proteins. Lane 1: MIL5scFv; lane 2: MIL5scFv-Arg9; Lane 3: Protein marker. D: Western blot analysis of purified proteins. Lane 1: MIL5scFv; lane 2: MIL5scFv-Arg9. The predicted molecular weight of MIL5scFv-Arg9 was about 49 kDa, which was almost the same with that of MIL5scFv. E: Flow cytometry analysis of antigen-binding capacity of purified proteins. Proteins were labelled with FITC in our lab. The F/P quotient of MIL5scFv-FITC is 4.0, while MIL5scFv-Arg9-FITC is 2.7. The cells were treated with labelled proteins on ice for 30 minutes and detected by flow cytometry, respectively. 1: Cells treated with herceptin-FITC as positive control; 2: Cells treated with MIL5scFv-FITC; 3: Cells treated with MIL5scFv-Arg9-FITC. MIL5scFv-FITC and MIL5scFv-Arg9-FITC was diluted as indicated (1:2, 1:10 or 1:50). The binding activity was evaluated with MFI value marked on the right top of each panel and the results showed that they could bind membrane antigen HER2 in a dose-dependent manner. MFI: mean fluorescence intensity. Data are representative of two independent experiments.