Biologic duoflow 10

PA cyt c₅₅₁ precipitates were produced by an addition of 80% (v/v) ethanol to 1 mM oxidized WT or M61A PA cyt c₅₅₁. The precipitate was separated from the supernatant by centrifugation, and lyophilized to remove residual ethanol. The obtained precipitate was dissolved with 1 ml of 50 mM potassium phosphate buffer, pH 7.0, at 4°C. Oligomer formation of PA cyt c₅₅₁ was analyzed by gel chromatography (Superdex 75, GE healthcare) using a fast protein liquid chromatography (FPLC) system (BioLogic DuoFlow 10, Bio-Rad, CA) at 4°C. WT PA cyt c₅₅₁ dimer was purified by repeating gel chromatography (HiLoad 26/60 Superdex 75, GE healthcare) using the FPLC system (BioLogic DuoFlow 10, Bio-Rad) with 50 mM potassium phosphate buffer, pH 7.0. Purified PA cyt c₅₅₁ dimer was used immediately after purification.

Multimer‐expressing HEK293 cells were cultured in FBS‐free Opti‐MEM medium (Life Technologies Europe BV). Supernatants were purified using BioLogic DuoFlow 10 medium‐pressure liquid chromatography system (FPLC; Bio‐Rad Laboratories NV) connected to Nickel His‐Trap 5 mL columns (GE Healthcare, VWR, Leuven, Belgium). Elution was performed using 20 mm phosphate buffer, pH 7.4 that contained 500 mm of sodium chloride (NaCl) and 500 mm of imidazole (Sigma‐Aldrich). Purified multimers were concentrated on Amicon Ultra 15, 50 K MWCO (Millipore‐Merck Chemicals NV/SA, Evere, Belgium), dialysed against PBS using Slide‐A‐Lyzer dialysis cassettes with 20K MWCO (Thermo Fisher Scientific) and quantified using NanoDrop 1000 (Thermo Fisher Scientific). To further enrich in high‐FHR4 valence molecular species, trifunctional FHR4‐heteromultimers were purified using an improved protocol including a first washing step of 50 mm imidazole followed by 125 mm imidazole to partly eliminate the molecular species with low‐FHR4 valence. A final two‐step elution was then performed using 1 m imidazole introducing a 2 h‐stop/resume in‐between (Fig. S3). Collected fractions from steps with 1 m imidazole peak 1 and peak 2 were pooled as fractions (f2) and 3 (f3), respectively. The fractions were concentrated, dialysed and quantified as described above in this section.

The water content of the apoferritin hydrogel was investigated by comparing the weights of dried and reswelled hydrogels. The hydrogels were dried by incubation at 50°C in a drying oven for 3 h. The stability of the apoferritin hydrogel was investigated by incubation in 1 M HCl, 1 M NaOH, 2-mercaptoethanol (2-ME), methanol, 43 μM trypsin, and 6 M guanidine hydrochloride (Gdn-HCl) at room temperature. The thermal stability of the apoferritin hydrogel was investigated by incubating the hydrogel in pure water at 20–100°C for 30 min. After the apoferritin hydrogel was heated at 90°C for 30 min, the heat-decomposed solution of the hydrogel was investigated by SEC (Superdex 200 increase 10/300 GL, GE Healthcare) using the FPLC system (Biologic DuoFlow 10, Bio-Rad) with 50 mM potassium phosphate buffer, pH 7.0, at 4°C, and the absorbance was monitored at 280 nm. The heat-decomposed solution of the hydrogel was also investigated by MALDI-TOF MS (Autoflex II, Bruker) using sinapinic acid as a matrix.