Synthesis of Ubiquitin Thioester Conjugates

Ub variants with the thioester group (Ub-SR) at the C-terminal G76 were generated from respective Ub monomers (unlabeled or ¹⁵N labeled) using the following procedure. A 1 mL solution of 5 mg/mL Ub was prepared in 20 mM sodium phosphate buffer (pH 8.0) with 10 mM ATP, 10 mM MgCl₂, 100 mM sodium 2-mercaptoethane-sulphonate (MESNA), and 250 nM of Ub-activating enzyme E1. ^{31 (link)} The mixture was incubated at 37°C for 6 hours and then stored at 4°C. The E1 enzyme was precipitated by adding a drop of glacial acetic acid to the solution. The protein solution was buffer-exchanged into dH₂O containing 0.4% TFA and subsequently lyophilized. Ub-SR generation was confirmed by ESI-MS (electrospray ionization mass spectrometry).