Histrap ff 5 ml column

For Ufd1 and Npl4 expression^{36 (link)}, each plasmid was transformed into Rosetta (DE3) Escherichia coli cells and grown at 37 °C in TB medium until they reached an OD₆₀₀ = 0.8. Protein expression was induced by addition of 0.5 mM IPTG at 18 °C overnight (for Npl4) or at 37 °C for 4 h (for Ufd1). Cells were harvested and resuspended in wash buffer (50 mM Hepes, 150 mM KCl, 3 mM MgCl₂, 5% glycerol, 25 mM imidazole (Sigma-Aldrich, 56750), pH 7.4). Resuspended cells were mixed (Ufd1 + Npl4), incubated for 30 min with lysozyme (PanReac AppliChem, A3711), lysed by sonication (5 × 30 s at 60% intensity) and centrifuged at 20,000 × g for 45 min. The supernatant was filtered, loaded onto a HisTrap FF 5 ml column (Cytiva), and washed with 40 column volumes of wash buffer. Proteins were eluted with NiNTA elution buffer (50 mM Hepes, 150 mM KCl, 3 mM MgCl₂, 5% glycerol, 300 mM imidazole, pH 7.4) and further purified by size-exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column equilibrated in gel filtration buffer (50 mM Hepes, 150 mM KCl, 3 mM MgCl₂, 5% glycerol, 1 mM DTT, pH 7.4). All purification steps were carried out at 4 °C and proteins were concentrated using 10 kDa centrifugal concentrator (Vivaspin Turbo 15) before snap freezing in liquid nitrogen and stored at −80 °C.

SM14est (Genbank: DAC80635.1) was codon optimized for heterologous expression in Bacillus subtilis and produced as previously described (Jensen et al., 2010 (link)), with modification during construct design such that the native SM14est signal peptide was replaced by AmyL (FJ556804.1) from B. licheniformis and a hexa histidine-tag was incorporated at the C-terminus of the protein. The recombinant SM14est protein (29 kDa) was purified by Ni-affinity chromatography on a HisTrap FF 5 mL column (Cytiva) using the ÄKTA™ Pure system. A peristaltic pump (MiniPuls 3, Gilson^®) was used for the initial column wash and loading steps. The purified protein was desalted using Spectra/Por^® dialysis membrane (MWCO 12–14 kDa). The molar extinction coefficient of 39,880 M⁻¹ cm⁻¹ was predicted by ExPASy-ProtParam and was used to calculate the concentration of purified enzyme from the absorbance measured at 280 nm. The SM14est sample was visualized following SDS-PAGE of 2.5 μg of purified protein on a Tris-Glycine 4–20% gel (Bio-Rad Laboratories). Thermal denaturation experiments were conducted in triplicate with 10 μM of enzyme and analyzed by nano differential scanning fluorimetry (nanoDFS) using a temperature slope of 2°C min⁻¹ (Prometheus NT.48, Nano Temper).

Fluorescently labeled Ub5 was synthesized as described previously (Abdul Rehman et al., 2016 (link)). Briefly, Cys-Ub 1-75 (containing a Cys residue upstream of M1) was coupled to pre-formed Ub4. The cysteine residue of this proximal ubiquitin was then conjugated to IRDye-800CW (Li-Cor). Fluorescently labeled longer K48-linked chains (Ub_n) were synthesized using a reaction mix containing 2500 μM WT ubiquitin, 250 μM (N-term 6His)-Ub (K48R, K63C), 0.5 μM UBE1, 15 μM UBE2R1, 10 mM ATP, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 0.6 mM DTT with an overnight incubation at 30°C. Chains with successful incorporation of 6His-Ub (K48R, K63C) at their distal end were separated from WT chains using a HisTrap FF 5 mL column (Cytiva). Chains were then fractionated by size over a Superdex 200 16/60 column. Fractions containing chains between Ub7-Ub20 were pooled and concentrated and buffer exchanged into PBS. An approximate concentration was determined using the median chain length and the chains were then reacted with a 3-fold molar excess of IRDye-800CW (Li-Cor) for 3 h at 22°C (600 rpm). The reaction was quenched with 50 mM BME and excess dye removed by desalting with a Hiprep 26/10 desalting column (Cytiva) followed by size-exclusion chromatography with a Superdex 200 16/60 column (Cytiva).