Fig. 5: Targeted Lys acylation on protein substrates containing endogenous Lys residues.
From: Repurposing a plant peptide cyclase for targeted lysine acylation
a, Scheme of the KL-tag, which consists of the KL recognition site flanked by 3-aa GSG linkers. b, Targeted Lys acylation via insertion of the KL-tag N-terminally, internally and/or C-terminally into sfGFP. Reactions were run in 100 mM HEPES buffer, pH 8, containing 5 µM sfGFP, 100 µM biotin-RNGLH, 400 µM NiSO4 and 0.5 µM OaAEP1 for 2 h at 25 °C. Each attached biotin-RN label culminated in a mass shift of +497 Da (calc. +497 Da). Further reaction time points are shown in Extended Data Fig. 5. c, Labelling of a KL-tagged single-domain antibody heterodimer with biotin-RNGLH or TAMRA-RNGLH, as indicated. Reactions were run in 100 mM HEPES buffer, pH 8, containing 50 µM protein, 750 µM biotin-RNGLH or 1 mM TAMRA-RNGLH, 1 mM NiSO4 and 1 µM OaAEP1 for 4 h at 25 °C. Mass shifts: +biotin-RN, +497 Da (calc. +497 Da); +TAMRA-RN, +682 Da (calc. +683 Da). Further reaction time points are shown in Extended Data Fig. 6 and Supplementary Fig. 21. d, SDS–PAGE analysis of CTC.445.2d labelling with TAMRA-RNGLH. Reactions were run in 100 mM HEPES buffer, pH 8, containing 50 µM protein (either KL-tagged or a variant where the Lys–Leu sequence was mutated to Ala–Leu), 1 mM TAMRA-RNGLH, 1 mM NiSO4 and 1 µM OaAEP1 for the indicated time points at 25 °C. The samples run on SDS–PAGE were also analysed by ESI-MS (Extended Data Fig. 7). e, Triple labelling of CTC-445.2d. In the first step, OaAEP1 was used to attach a C-terminal alkyne click handle via incorporation of propargylamine. Next, sortase A was used to label the protein N terminus with TAMRA-LPET. Finally, the KL-tag was acylated with biotin-RN in a second OaAEP1-catalysed step. For all panels, the spectra shown are reconstructed ESI-MS spectra. The peptide label:protein ratios were calculated based on relative peak heights and are shown adjacent to the spectra.
