Enzymatic Assembly of Polyubiquitin Chains

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.