Superdex 75 26 60

B56γ cell pellets were resuspended in ice-cold lysis buffer (50 mM Tris pH 8.0, 0.5 M NaCl, 5 mM imidazole, 0.1% Triton X-100 containing EDTA-free protease inhibitor tablet [Sigma]), lysed by high-pressure cell homogenization (Avestin C3 Emulsiflex) and centrifuged (35,000 xg, 40 min, 4°C). The supernatant was loaded onto a HisTrap HP column (GE Healthcare) pre-equilibrated with Buffer A (50 mM Tris pH 8.0, 500 mM NaCl and 5 mM imidazole) and was eluted using a linear gradient of Buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Fractions containing the protein were pooled and dialyzed overnight at 4°C (50 mM Tris pH 8.0, 500 mM NaCl) with TEV protease to cleave the His₆-tag. The cleaved protein was incubated with Ni²⁺-NTA beads (GEHealthcare) and the flow-through collected. The protein was concentrated and purified using size exclusion chromatography (SEC; Superdex 75 26/60 [GE Healthcare]) pre-equilibrated in ITC buffer (50 mM sodium phosphate pH 7.5, 150 mM NaCl, 0.5 mM TCEP) or crystallization buffer (20 mM HEPES pH 7.8, 500 mM NaCl, 0.5 mM TCEP). Fractions were pooled, concentrated to designated concentration for experiments or stored at −80 °C. KIF4A_1192-1232 was purified similarly except that it was heated at 80°C for 10 min and centrifuged (15,000 xg, 10 min, 4°C) prior to SEC purification (SEC buffer: 20 mM HEPES pH 7.8, 500 mM NaCl, 0.5 mM TCEP).

Immediately prior to the ITC experiments, SEC (Superdex 75 26/60, GE Healthcare) was used to transfer BdcA into assay buffer followed by concentration of the BdcA protein. Ligands were also dissolved in assay buffer, and both protein and ligand were degassed under vacuum. ITC experiments were performed using a VP-ITC (GE Healthcare) at 25°C. Ligands (600 µM) were titrated (10 µL injections every 200 s, 35 times for all runs except c-di-GMP, in which the time between injections was 300 s) into the cell containing 12 µM monomeric BdcA under constant stirring (307 rpm). Titration of the ligand into buffer alone resulted in a negligible heat of dilution for each ligand. The active protein concentration was adjusted by fitting the data to a one-site binding model. Association constant (K_a) values were calculated using Microcal Origin 7.0 software.

All proteins were expressed in Escherichia coli BL21(DE3) cells, grown to an OD₆₀₀ of 0.8 and induced with 0.3 mM IPTG overnight at 16 °C. Hsp27 and its variants were purified with HisTrapFF column (GE Healthcare) with the Tris buffer (50 mM Tris-HCl, 100 mM NaCl, a gradient of 0~500 mM imidazole, pH 8.0). The N-terminal His₆-tag was removed using TEV protease in the buffer of 50 mM Tris-HCl, 100 mM NaCl, pH 8.0. The cleaved proteins were immediately loaded onto the size-exclusion chromatography column Superdex 75 26/60 (GE Healthcare) with a PBS buffer of 50 mM sodium phosphate, 50 mM NaCl at pH 7.0.
Human Tau40 and K19 were over-expressed and purified as previously described (Barghorn et al., 2005 (link)). Briefly, Tau/K19 was purified by a HighTrap HP SP (5 ml) column (GE Healthcare), and followed by a Superdex 75 gel filtration column (GE Healthcare).
For ¹⁵N-labeled proteins, protein expression was the same as that for unlabeled proteins except that the cells were grown in M9 minimal medium with ¹⁵NH₄Cl (1 g l⁻¹). Purification of ¹⁵N-labeled proteins was the same as that of the unlabeled proteins.
The purity of proteins was assessed by SDS-PAGE. Protein concentration was determined by BCA assay (Thermo Fisher).

The human PXR-LBD (130-434) was co-produced with a fragment of the steroid receptor coactivator-1 (SRC-1, 623-710) to enhance PXR stability. The PXR-LBD gene was cloned into pRSET-A with a His₆ tag at the N-terminus (gift from Matthew Redinbo, University of North Carolina at Chapel Hill, USA), and the SRC-1 fragment gene has been inserted into the pACYC184 vector. Proteins were overproduced in E. coli BL21(DE3) cells overnight at 20 °C in LB medium without any ligand. After culture cells were harvested by centrifugation and the pellets resuspended in lysis buffer (20 mM Tris pH 7.5, 250 mM NaCl, 5% (v/v) glycerol) supplemented with lysozyme (1 μg ml⁻¹) and a protease inhibitor cocktail tablet (cOmplete, EDTA-free, Roche), and then subjected to sonication. The clarified cell lysate was applied onto a Ni²⁺-affinity column (HisTrap 5 ml; GE Healthcare) equilibrated with lysis buffer supplemented with 10 mM imidazole. The eluted PXR-LBD was then applied onto a gel filtration column (Superdex 75 26/60; GE Healthcare) equilibrated with a buffer containing 20 mM Tris pH 7.8, 250 mM NaCl, 5% (v/v) glycerol, 5 mM DTT, 1 mM EDTA. The PXR-LBD was concentrated and stored at −40 °C.

α-Synuclein protein expression, purification and oligomer formation were carried out as described previously^{24 (link)}. Briefly, α-synuclein was purified from Escherichia coli BL21 cells overexpressing α-synuclein, which were lysed by sonication. Heat-sensitive proteins were precipitated out of the lysate supernatant by boiling. Following centrifugation, streptomycin sulphate was added to the supernatant and centrifuged again. α-Synuclein was precipitated out of the supernatant by addition of ammonium sulphate (361 mg ml⁻¹) and stirring at 4 °C for 30 min. The precipitated α-synuclein was resuspended 25 mM Tris buffer, pH 7.4, 20 °C and loaded onto a HiLoad 26/10 Q Sepharose high performance column (GE Healthcare Ltd., Little Chalfont, UK), and eluted at ~ 350 mM NaCl, 20 °C with a salt gradient from 0 to 1.5 M NaCl. Selected fractions were subsequently loaded onto a Superdex 75 26/60 (GE Healthcare Ltd.) at 20 °C and eluted in PBS, pH 7.4, 20 °C. Protein concentration was determined by absorbance at 275 nm, using an extinction coefficient of 5600 M⁻¹ cm⁻¹.

The dual expression plasmid was transformed into Rosetta II (DE3) (Invitrogen) E. coli strain, grown in Luria Bertani medium, and induced with 1 mM isopropyl-β-d-thiogalactopyranoside at an A₆₀₀ ~ 0.8 for 2 hours. The bacterial pellets from 1L of growth media were resuspended in 30 mL of B-PER reagent (Thermo Scientific) and expressed protein purified from the lysis supernatant by Nickel affinity and gel filtration (Superdex 75 26/60, GE Healthcare) chromatography. The purified protein was concentrated and passed over the gel filtration column a second time before Western blot analysis.