The construct pTYB-HAUb, comprising the sequences of the human Ub (lacking Gly 76), an intein and a chitin binding domain, plus an HA tag, was used to synthesize HAUb75-MESNa as described previously (Borodovsky et al., 2002 (link)). Briefly, Ub–intein–chitin domain fusion protein was expressed in Escherichia coli (18 h induction with 0.4 mM IPTG at 17°C). Cell pellets were resuspended in 50 mM HEPES, pH 7.4, 150 mM NaCl, and 0.5 mM TCEP and lysed in a high-pressure homogenizer. The cleared cell extract was loaded onto a 15 ml chitin bead (New England Biolabs) column at a flow rate of 0.5 ml/min. The column was washed with 60 ml of lysis buffer followed by 25 ml of lysis buffer containing 50 mM β-mercaptoethanesulfonic acid sodium salt (MESNa) and incubated overnight at 37°C for the induction of on-column cleavage. HAUb75-MESNa thioester was eluted with 25 ml of lysis buffer and concentrated: approximately 2.5 mg of protein was recovered from a 1-L culture. The N-terminal Met of the HA-tag was frequently processed off during expression, resulting in a mixture of two proteins that behaved identically in labeling experiments.
To synthesize the HA-UbC2Br or HA-UbPA probes, 0.2 mM of 2-bromoethylamine or 250 mM propargylamine was added to a solution of HAUb75-MESNa (1–2 mg/ml) in 500 μl of column buffer, respectively. pH was carefully adjusted to 8 with NaOH, and after 20 min shaking at 1,400 rpm, at room temperature, 100 μl of 2.0 M aqueous HCl was added and the resultant reaction mixture was promptly transferred to a PD10 gravity column for buffer exchange, according to the manufacturer's instructions.
The probe was then aliquoted and frozen at −80°C for storage (no significant deterioration is observed for several months of storage except for HA-UbC2Br, which is prone to hydrolysis). All HA-Ub-derived probes were analyzed by liquid chromatography mass spectrometry (LC-MS) using a 1290 UPLC (Agilent) coupled to a 6560 quadrupole time-of-flight (QToF) mass spectrometer (Agilent) to monitor the reaction and the product detected by [M+H]+ = 10,197.6221, with >90% purity.
To synthesize the HA-UbC2Br or HA-UbPA probes, 0.2 mM of 2-bromoethylamine or 250 mM propargylamine was added to a solution of HAUb75-MESNa (1–2 mg/ml) in 500 μl of column buffer, respectively. pH was carefully adjusted to 8 with NaOH, and after 20 min shaking at 1,400 rpm, at room temperature, 100 μl of 2.0 M aqueous HCl was added and the resultant reaction mixture was promptly transferred to a PD10 gravity column for buffer exchange, according to the manufacturer's instructions.
The probe was then aliquoted and frozen at −80°C for storage (no significant deterioration is observed for several months of storage except for HA-UbC2Br, which is prone to hydrolysis). All HA-Ub-derived probes were analyzed by liquid chromatography mass spectrometry (LC-MS) using a 1290 UPLC (Agilent) coupled to a 6560 quadrupole time-of-flight (QToF) mass spectrometer (Agilent) to monitor the reaction and the product detected by [M+H]+ = 10,197.6221, with >90% purity.
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