Fig. 1: Genetic HSA variants with modified C-terminal ends show reduced FcRn binding.

a An overview of the C-terminal sequences of genetic HSA variants. b A 12% SDS-PAGE gel stained with Coomassie Blue showing 2 μg of purified recombinant WT HSA and genetic variants. The gel was cropped and the full-length gel is shown in Supplementary Fig. 1. c Relative expression levels of WT HSA and genetic variants by transiently transfected HEK293E cells. Protein secreted into the growth medium was quantified by ELISA, and the values represent the mean ± s.d. of four replicates. d SPR sensorgrams showing binding of titrated amounts (0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, and 16 μM) of monomeric hFcRn injected over immobilized (~200 RU) WT HSA at pH 5.5 (—) and the fit of the data to the 1:1 binding model (·····). Injections were performed at 25 °C, and the flow rate was 40 μl/min. e–h SPR sensorgrams showing binding of titrated amounts (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 μM) of monomeric hFcRn injected over immobilized (~500 RU) (e) Bazzano, (f) Catania, (g) Rugby Park, and (h) Venezia at pH 5.5. Injections were performed at 25 °C, and the flow rate was 10 μl/min. (i) An illustration of the co-crystal structure of WT HSA in complex with hFcRn. DI, DII, and DIII of HSA are colored in pink, wheat, and light blue, respectively. The three domains, α1, α2, and α3, of the HC of hFcRn are shown in light green and the β₂m subunit in gray. j A close-up of the two last α-helices of the C-terminal end of HSA. The figures were made using PyMOL with the crystallographic data of WT HSA-hFcRn (PDB ID 4N0F)²⁶.