Fig. 2

Assembly of SA-FGF1 oligomers. A BirA-mediated site-specific FGF1-AviTag biotinylation. FGF1 was fused to the AviTag acceptor peptide and this Avi-tagged protein was selectively biotinylated by GST-BirA enzyme, leading to the formation of site-specific mono-biotinylated ligand. Attached biotin is marked in yellow. B and C GST-BirA was purified by glutathione affinity chromatography. Protein purity was analyzed by SDS–PAGE (CBB staining) (B) and western blotting (WB) with anti-GST antibody (C). D Biotinylation of FGF1-AviTag. The efficiency of the biotinylation (seen as upshift in protein migration) and purity of FGF1-AviTag-Biot was analyzed by SDS-PAGE (CBB staining) E Mass spectrometry analysis of purified FGF1-AviTag-Biot. The molecular mass of FGF1-AviTag-biot was assessed by MALDI MS. The theoretical MW of the proteinaceous core of FGF1-AviTag is 17092 Da, and 17,318 Da after biotinylation. F–J BLI comparison of FGF1-AviTag and FGF1-AviTag-Biot interaction with various SA variants. FGF1-AviTag and FGF1-AviTag-Biot were chemically immobilized on BLI sensors and incubated with SA-4D (F), SA-1A3D (G), SA-2A2D (H), SA-3A1D (I), SA-4D (J). The association and dissociation profiles were measured. K and L Assembling of FGF1-AT-Biot-SA-4A oligomer. FGF1E-AviTag and biotinylated variant of this protein 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 and CBB staining (K) and western blotting (L); *most likely self-assembly form of FGF1-AviTag or partial SA dissociation product upon SDS treatment. M–O Assembling of various FGF1-SA oligomers. Protein components were mixed in 1:1 ratio and incubated for 5–10 min at RT. Then, protein mixes were subjected to SDS-PAGE analysis and CBB staining