Fig. 1

Basic building blocks for development of SA-FGF1 oligomers. A Strategy for development of FGF1-SA oligomers. In this approach SA tetramers containing from 0 to 4 binding sites for biotinylated ligands are obtained by mixing wild type SA-Alive-HisTag and non-biotin-binding SA-Dead mutant. FGF1-AviTag will be enzymatically biotinylated by GST-BirA and then assembled with various SA scaffolds, leading to formation of FGF1-SA oligomers with distinct potential for clustering of FGFR1. B and C Separately purified SA-Alive-HisTag and non-biotin-binding SA-Dead. The purity and identity of streptavidin variants were confirmed by SDS-PAGE (CBB staining), presence of the HisTag on the SA-Alive version allows for comparison of SA variants composition. To maintain the tetrameric form of the protein, SA samples in C were not subjected to thermal denaturation. D and E Variants of streptavidin tetramers with varying valency. Separately purified SA-Alive-HisTag and SA-Dead were mixed, yielding all possible combinations. Due to the presence of the His-Tag on SA-Alive version (ensuring affinity to metal ions), metal-affinity chromatography was applied to separate various combinations of SA tetramers. The purity and identity of obtained SA variant were confirmed by SDS-PAGE. Upon boiling, samples were separated into monomers (D). The ratio of two SA-bands demonstrates protein tetramers containing from 0 to 4 binding sites for biotinylated ligands. To preserve the tetrameric form of SA, samples in E were not subjected to thermal denaturation. F and G FGF1-AviTag protein was purified by heparin affinity chromatography. Purified protein was analyzed by SDS–PAGE under reducing condition (F) and western blotting (WB) with antibody directed against FGF1 (G). H CD spectra of wild type FGF1 and FGF1-AviTag confirming preservation of 2D structure of FGF1 upon incorporation of AviTag. I BLI comparison of FGF1 and FGF1-AviTag interaction with FGFR1. The extracellular region of FGFR1 was immobilized on BLI sensors and incubated either with FGF1 and FGF1-AviTag. The association and dissociation profiles were measured. J FGF1-AviTag is able to activate FGFR1. Serum-starved NIH3T3 cells were incubated for 15 min with different concentrations of FGF1 (positive control) or FGF1-AviTag in the presence of heparin. Cells were lysed and activation of FGFR1 assessed with western blotting using antibodies recognizing phosphorylated key tyrosine within intracellular FGFR tyrosine kinase domain (pFGFR) and receptor-downstream ERK (detected with antibodies recognizing phosphorylated ERK (pERK). The level of tubulin served as a loading control