Fig. 5
Development of tetrameric FGF1-SA. A Strategy for generation of the cytotoxic FGF1-SA oligomer. FGF1E-AviTag was conjugated to the cytotoxic compound MMAE via N-terminal cysteine flanked by two lysines. The conjugated protein was then biotinylated and assembled with tetrameric SA-4A to yield a cytotoxic tetrameric conjugate. Conjugated N-terminal cysteine is marked in blue, attached cytotoxic molecules are marked in red and attached biotin is marked in yellow. B and C FGF1E-AviTag protein was purified by heparin affinity chromatography. Using SDS-PAGE (B) and western blotting (C) with antibodies directed against FGF1, the purity and identity of protein were verified. D FGF1E-AviTag-Biot is able to activate FGFR1. Serum-starved NIH3T3 cells were incubated for 15 min with FGF1 (positive control) or with different concentrations of FGF1E-AviTag-Biot with the presence of heparin. Cells were lysed and activation of FGFR1 (pFGFR), and receptor-downstream signaling (pERK) was assessed with western blotting. The signal of non-modified FGFR and ERK served as a loading control. E Conjugation of FGF1E-AviTag with cytotoxic MMAE. The efficiency of the conjugation and biotinylation and purity of obtained MMAE-FGF1E-AviTag-Biot were confirmed by SDS-PAGE and CBB staining. F BLI comparison of MMAE-FGF1E-AviTag and MMAE-FGF1E-AviTag-Biot interaction with SA-4A. Both conjugates were chemically immobilized on BLI sensors and incubated with SA-4A. The association and dissociation profiles were measured. G Assembling of MMAE-FGF1E-AviTag-Biot-SA-4A oligomer. MMAE-FGF1E-AviTag and MMAE-FGF1E-AviTag-Biot were mixed with SA-4A in various ratios and incubated for 5–10 min at RT. Then, proteins mixes were subjected to SDS-PAGE analysis. H Size exclusion chromatography of MMAE-FGF1E-AviTag-Biot-SA-4A oligomer. The absorbance spectra were monitored at 280 nm. I. and J. The efficiency of the folding and purity of MMAE-FGF1E-AviTag-Biot-SA-4A were analyzed with SDS-PAGE (I) and western blotting with antibodies recognizing FGF1 (J). To maintain the tetrameric form of the protein, samples were not subjected to thermal denaturation. K BLI-determined kinetic parameters of the interaction between MMAE-FGF1E-AviTag-Biot-SA-4A and FGFR1. The extracellular region of FGFR1 was immobilized on BLI sensors and incubated with various concentrations of MMAE-FGF1E-AviTag-Biot-SA-4A. KD, kon, and koff were calculated using global fitting with the 2:1 “heterogeneous ligand” with ForteBio Data Analysis 11.0 software