Monos 10 100 gl column

Polyubiquitin chains were assembled enzymatically by combining monoubiquitin (>1 mM), human UBE1 enzyme (500 nM), and Saccharomyces cerevisiae Ubc13/Mms2 (2.5 μM) in a solution containing 50 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM TCEP, and 10 mM ATP. To limit the chain length to diubiquitin, K48R/K63R ubiquitin and D77 ubiquitin can be substituted for WT ubiquitin in the reaction mixture (19 (link)). K48R/K63R ubiquitin will occupy the distal position in K63Ub₂, and D77 ubiquitin will occupy proximal position in K63Ub₂. Human UBE1 and S. cerevisiae Ubc13/Mms2 enzymes were expressed and purified, as previously described (20 (link), 21 (link)). The reaction mixture was incubated overnight at 37 °C and then diluted 10-fold in buffer A (50 mM ammonium acetate pH 4.5 and 50 mM NaCl) and loaded onto a monoS 10/100 GL column (Cytiva life sciences) equilibrated in buffer A. The ubiquitin species retained by the column were eluted by running a gradient from 0 to 100% buffer B (50 mM ammonium acetate pH 4.5 and 600 mM NaCl) over 300 ml.

Total bacterial DNA was isolated by using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Primers were designed using the coding sequence (CDS) of locus tag LLG50_11005 of the G50 genome sequence (accession number CP025500.1) (S1 Table).
DNA fragments encoding variants of YwfG (residues 28–270, 28–336, 28–511, and 860–1034) were amplified by polymerase chain reaction using the primer sets Fw28–Rv270, Fw28–Rv336, Fw28–Rv511, and Fw860–Rv1034, respectively. The resulting fragments were inserted in NdeI/BamHI-digested pET28b (Merck) by In-Fusion cloning reactions (In-Fusion HD Cloning Kit, Takara Bio, Kusatsu, Japan) with an N-terminal His₆-tag. The resulting constructs were transformed into E. coli BL21(DE3). Recombinant YwfG variants were expressed at 16°C overnight in the presence of 0.2 mM isopropyl β-d-thiogalactoside and purified by affinity chromatography using a nickel-chelating column (HisTrap HP, Cytiva), followed by gel filtration using a HiLoad 26/600 Superdex 75 pg column (Cytiva) and cation exchange chromatography using a Mono S 10/100 GL column (Cytiva). The obtained recombinant YwfG variants were named YwfG_28–270, YwfG_28–336, YwfG_28–511 (numbers indicate amino acid residues), and MubR4 (residues 860–1034).