Pet 50b derivative plasmid

Chemical reagents were purchased from Sigma-Aldrich unless indicated otherwise. A pET-50b-derivative plasmid harboring a synthetic gene of human ubiquitin was purchased from GenScript. Escherichia coli BL21(DE3) cells were transformed with this plasmid and were cultured at 37 °C in minimal media containing 1 g/L ¹⁵NH₄Cl (Cambridge Isotope Laboratories) as the sole nitrogen source in the presence of 30 μg/L kanamycin. When the optical density at 600 nm reached 0.8 for the culture, 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce expression of ubiquitin. The culture was continued at 18 °C for 16 h. Ubiquitin (¹⁵N-labeled) was purified through the procedures of Sundd et al.^{27 (link)} and additionally, through size-exclusion chromatography using a Sephacryl S-100 column (GE Healthcare) equilibrated by a buffer of 100 mM ammonium acetate at pH 7.0. The purified ¹⁵N ubiquitin was lyophilized and kept at −20 °C until use.

Chemical reagents were purchased from Sigma-Aldrich unless indicated otherwise. A pET-50b derivative plasmid that contains a synthetic sequence encoding human ubiquitin at the NdeI and XhoI sites was purchased from GenScript. Escherichia coli strain BL21(DE3) was transformed with this plasmid and cultured at 37°C in minimal media containing 4 g/L ¹³C glucose (Cambridge Isotope Laboratories) and 1 g/L ¹⁵NH₄Cl (Cambridge Isotope Laboratories) as sole carbon and nitrogen sources in the presence of 30 μg/L kanamycin. After induction of protein expression by 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), the culture was continued at 18°C for 16 hours. ¹³C,¹⁵N-labeled ubiquitin was purified from the lysate through the procedures of Sundd et al.^{32 (link)} The protein was further purified through a Sephacryl S-100 size-exclusion column (GE Healthcare) equilibrated by a buffer of 100 mM ammonium acetate at pH 7. The purified ubiquitin was lyophilized and kept at −20°C until use.