Superdex 75 gel filtration column

The plasmids pET28aNS-CaBglA, pET28aNS-CtBglA, pET28aNS-CglT, and pET28aNS-Td2f2 were transformed into E. coli BL21(DE3) for heterologous expression of CaBglA, CtBglA, CglT, and Td2f2, respectively. Synthesis of recombinant proteins in E. coli BL21(DE3) cells was initiated by the addition of 1 mM IPTG, and cultivation was continued for an additional 16 h at 16 °C. Cells were harvested by centrifugation at 10,000 rpm, resuspended in 50 mM Tris–HCl buffer containing 30 mM imidazole and 300 mM NaCl, pH 8.0, and lysed by ultrasonication. The supernatants were applied onto a Histrap™ HP Ni-affinity column (GE Healthcare). The proteins were eluted with 50 mM Tris–HCl buffer containing 500 mM imidazole and 300 mM NaCl, pH 8.0. The eluted fractions were then concentrated to 2 mL using Amicon Ultra-15 centrifugal filter units (10.0 kDa cutoff) (Merck Millipore, Billerica, MA, USA), and applied onto a Superdex 75 gel filtration column (GE Healthcare) with 50 mM K₂HPO₄–KH₂PO₄ buffer with 100 mM KCl, pH 6.0.

Both laccase variants were deglycosylated using 1000 U mg⁻¹ endoglycosidase Hf (New England BioLabs, USA) and 0.1 U mg⁻¹ α-mannosidase (Sigma, USA) in 50 mM sodium citrate buffer pH 5.5 containing 10 mM ZnCl₂ for 3 h at room temperature. The deglycosylation mixture was loaded onto a Superdex 75 gel-filtration column (GE Healthcare, USA) equilibrated with 50 mM sodium citrate buffer pH 5.5 to remove the deglycosylating enzymes. Pure fractions were concentrated and stored at 4°C. The deglycosylated forms of BaL and L499M BaL were only used for crystallization experiments.

The MBP tag and coding sequence of FIONA1 were cloned into PET28a vector containing His tag for protein expression and purification. The MBP tag can promote the expression and stability of the recombinant protein. The recombinant plasmid containing MBP-FIONA1-His was transfected into E. coli strain BL-21 Gold competent cells. The E. coli cells were grown at 37 °C to an OD600 of 0.6–0.8, and recombinant protein expression was then induced at 18 °C with 500 nM IPTG for 20 h. Then the pellet from each 2 L culture was collected, resuspended in 30 mL of lysis buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM PMSF, 3 mM DTT, 5% glycerol), and sonicated for 10 min. The sample was centrifuged at 13,000 rpm for 30 min, and the supernatant was filtered through a 0.22-μm filter (Millipore), then loaded on a Ni-NTA column (GE Healthcare). After washing in 20 ml Buffer A (10 mM Tris pH 7.9, 150 mM NaCl) and then 20 ml 8% Buffer B (10 mM Tris-HCl pH 7.9, 150 mM NaCl, 500 mM imidazole), protein was eluted by Buffer B. The collected fraction was then purified by a Superdex 75 gel-filtration column (GE Healthcare, 10 mM Tris-HCl pH 7.9, 150 mM NaCl, and 3 mM DTT. Protein was concentrated into 17.8 mg ml^− 1 and 20% glycol was added. Aliquots of protein were frozen by liquid N₂ then stored in − 80 °C.

The C-terminal StrepII-His₈ tagged MSMEG_3494_33–153 was expressed in E.coli BL21 (DE3) by periplasmic expression vectors pML4249 or pML4604 containing a E.coli signal sequence (Table S2). One liter of Luria–Bertani (LB) medium was used for producing the unlabeled protein, and two liters of M9 minimal medium containing 4 g ¹⁵NH₄Cl were used for producing ¹⁵N-labeled proteins. Cells were grown at 37 °C until the cell density reached an OD₆₀₀ of ~1.8. Then the cells were induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18 °C overnight. Afterward, cells were harvested by centrifugation and lysed by sonication in ice-cold lysis buffer (150 mM NaCl, 30 mM Tris, 1 mM PMSF [pH 7.5]). Cell lysate was clarified by centrifugation, and the supernatant flowed through Ni-NTA column. Approximately 20 mg of the protein was obtained by Ni affinity purification. Afterward, the affinity tag was removed by enterokinase, and a total of ~15 mg isotopically labeled protein was obtained after size exclusion chromatography via Superdex 75 gel filtration column (GE).

The Crl–σ^S binary complex was prepared by incubating Crl and σ^S at a molar ratio of 2:1 and purified by using a Superdex 75 gel filtration column (GE Healthcare). The E. coli Crl–TAC was assembled by directly incubating the RNAP core enzyme, the Crl–σ^S binary complex, and the nucleic-acid scaffold at a molar ratio of 1:4:4 at 4°C overnight. The mixture was loaded onto a Superdex 200 gel filtration column (GE Healthcare) and eluted with 10 mM HEPES [pH 7.5], 50 mM KCl, 5 mM MgCl₂, and 3 mM DTT. Fractions containing E. coli Crl–TAC were collected and concentrated to ~12 mg/ml.

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).

To study complex formation between the Rab proteins and C9orf72, 3× molar excess of the each Rab protein was mixed with C9orf72 and incubated overnight on ice in the presence of 20 mM EDTA. The protein mixture was injected onto a Superdex75 gel-filtration column (GE Healthcare, Buckinghamshire, UK) equilibrated with buffer containing 20 mM Tris (pH 8.0), 300 mM NaCl, two mM DTT and 20 mM EDTA. The sample was applied to the column using a 100 μl loop at 0.5 ml/min using an AKTA PrimePlus FPLC system (GE Healthcare, Buckinghamshire, UK). Elution profile was monitored by UV absorbance at 280 nm. These were compared to injections of Rab proteins alone. Fractions were analyzed by reducing analytical SDS–PAGE using Coomassie Blue staining and western blot.