FGF1V solution (30 µM) in 25 mM phosphate buffer, pH 7.4, and 100 mM NaCl was reduced with 1 mM TCEP for 20 minutes at room temperature, desalted with a Zeba spin column (Thermo Fisher Scientific, Waltham, MA, USA), and added to a CH3CN solution of linker-functionalized MMAE (vcMMAE) containing a maleimide moiety, and the conjugation was carried out at 4°C. There was a two- to fivefold molar excess of the drug over the FGF1V N-terminal –SH group. The reaction was quenched after 16 hours with an excess of free cysteine. Different reaction conditions and durations were tested in order to achieve optimum conjugation efficiency with protein structure and function retained. Reaction progress was monitored by SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
To purify the conjugate, unmodified FGF1V was removed by hydrophobic interaction chromatography on phenyl-Sepharose (GE Healthcare, Chicago, IL, USA). The conjugation reaction mixture was loaded on a phenyl-Sepharose column equilibrated in 25 mM Tris-HCl, pH 7.4, and 2 M NaCl, and FGF1V–vcMMAE was eluted with a linear gradient of decreasing salt concentration (from 0% to 100% of 25 mM Tris-HCl, pH 7.4, 0.1 M NaCl). Identity and purity of conjugated FGF1V–vcMMAE were confirmed by SDS-PAGE, Western blotting, and MALDI-TOF MS.
To purify the conjugate, unmodified FGF1V was removed by hydrophobic interaction chromatography on phenyl-Sepharose (GE Healthcare, Chicago, IL, USA). The conjugation reaction mixture was loaded on a phenyl-Sepharose column equilibrated in 25 mM Tris-HCl, pH 7.4, and 2 M NaCl, and FGF1V–vcMMAE was eluted with a linear gradient of decreasing salt concentration (from 0% to 100% of 25 mM Tris-HCl, pH 7.4, 0.1 M NaCl). Identity and purity of conjugated FGF1V–vcMMAE were confirmed by SDS-PAGE, Western blotting, and MALDI-TOF MS.