Protein expression and purification of metK bearing proteins

The proteins sfGFP(N150TAG)-His₆, myoglobine(S4TAG)-His₆, Rab1b(Q76A, R79TAG)-His₆, and GFP-nanobody (R18TAG)-His₆ were expressed overnight at 37 °C in 2xYT with the respective antibiotics. At OD 0.6–0.7, protein expression was induced using 0.02% (w/v) arabinose. The medium was further complemented with 1 mM met-ncbK.⁸⁵

sfGFP and myoglobine were purified as described above. GFP-nanobody was also purified as described above using the respective wash and elution buffers (nanobody wash buffer: 1× PBS, 500 mM NaCl, 30 mM imidazole, 2 mM PMSF; nanobody elution buffer: 1× PBS, 500 mM NaCl, 300 mM imidazole). The same purification protocol was used for Rab1b, although the respective wash buffer was complemented with 0.2 mM PMSF, 0.01 mM GDP, cOmplete protease inhibitor cocktail tablets (Roche) for cell lysis (Rab1b wash buffer: 40 mM HEPES pH 8, 2 mM ß-ME, 1 mM MgCl₂, 500 mM LiCl, 0.01 mM GDP, 20 mM imidazole; Rab1b elution buffer: 40 mM HEPES pH 8, 2 mM ß-ME, 1 mM MgCl₂, 500 mM LiCl, 0.01 mM GDP, 300 mM imidazole).^85,87

The incorporation of met-ncbK and quantitative decaging to metK was confirmed using full-length ESI-LC MS (Phenomenex Jupiter^TM C4 column (2 × 150 mm, 5 μm)) and carried out on an Agilent Technologies 1260 Infinity LC–MS system with a 6310 quadrupole spectrometer. Buffer A was 0.1% formic acid in water; buffer B was 0.1% formic acid in acetonitrile.