Astra version 7

The full length HSF5 expression construct was cloned into a pMAL-c6T vector (New England Biolabs) and fused to His⁶-MBP and a tobacco etch virus protease cleavage site at the N-terminus. His-MBP-full length HSF5 was purified by HisTrap HP (Cytiva), and gel filtration using a Superdex 200 pg 16/600 column (Cytiva). Size exclusion chromatography with multi-angle light scattering (SEC–MALS) was performed using a DAWN HELEOS8+ (Wyatt Technology Corporation, Santa Barbara, CA, USA), a high-performance liquid chromatography pump LC-20AD (Shimadzu, Kyoto, Japan), a refractive index detector RID-20A (Shimadzu), and a UV–vis detector SPD-20A (Shimadzu), which were located downstream of the Shimadzu liquid chromatography system connected to a PROTEIN KW-803 gel filtration column (Cat. no. F6989103; Shodex, Tokyo, Japan). Differential RI (Shimadzu) downstream of MALS was used to determine the protein concentrations. The running buffer used contained 25 mM HEPES/KOH (pH 7.2) and 150 mM KCl. Approximately 100 μL of the sample was injected at a flow rate of 1.0 mL /min. Data was then analyzed using ASTRA version 7.0.1 (Wyatt Technology Corporation). Molar mass analysis was also performed over half of the width of the UV peak top height. 30 min after incubation at 42 °C, 100 μL of MBP-full length HSF5 (12.6 μM) or HSF5-DBD (26.6 μM) was injected.

SEC-MALS was measured using DAWN HELEOS8+ (Wyatt Technology Corporation) downstream of a Shimadzu liquid chromatography system connected to Superdex200 10/300 GL (GE Healthcare) gel filtration column. The differential refractive index (Shimadzu Corporation) downstream of MALS was used to obtain protein concentration. The column was equilibrated with a running buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl₂, 10% glycerol, and 2 mM β-mercaptoethanol. Flow rate was set to 0.5 mL min⁻¹ and 100 µL of the sample was injected. Kapβ2 at a concentration of 28 µM was injected in the absence and presence of 55 µM of MBP:PR18. The data were analyzed with ASTRA version 7.0.1 (Wyatt Technology Corporation).

Single-domain antibody samples were concentrated to 7.5 mg/mL and subject to ten freeze-thaw cycles by placing at minus 80°C (15 mins) followed by room temperature thawing (15 mins). Aggregation content was subsequently determined using an analytical SEC column (AdvanceBio SEC 130Å) fitted to a 1260 Infinity High-performance liquid chromatography (HPLC) system (Agilent) and coupled in series to a multi-angle static light scattering (MALS) detector and differential refractometer (Wyatt Technology). All samples were injected at a flow rate of 0.4 mL/min into a mobile phase comprising PBS at room temperature. The refractive index increment (dn/dc) on the MALS detector was set to 0.185 for protein analysis. Data was analysed using ASTRA (version 7; Wyatt Technology).

SEC-MALS was measured using the instrument consisting of a high-performance liquid chromatography (HPLC) pump LC-20AD (Shimadzu, Kyoto, Japan), a refractive index detector RID-20A (Shimadzu), a UV-vis detector SPD-20A (Shimadzu), a light scattering detector DAWN HELEOS8+ (Wyatt Technology Corporation, Santa Barbara, CA, USA), and a TSKgel G3000SW_XL column (Tosoh Bioscience, Tokyo, Japan) [for TtTF (EDTA) and TtTF (Zn²⁺)] or KW-803 column (Shodex, Tokyo, Japan) (for natively purified TtTF). The centrifuged (15,000 rpm for 5 min at 4 °C) TtTF (Zn²⁺) or TtTF (EDTA) samples at various concentrations in buffer containing 50 mM HEPES-KOH and 100 mM KCl at a pH of 7.5, were injected into the HPLC system at the 1 mL/min flow rate. Data were analyzed with ASTRA version 7 (Wyatt Technology Corporation).