Figure 2
From: Modular Protein Ligation: A New Paradigm as a Reagent Platform for Pre-Clinical Drug Discovery
Choice of intein impacts ligation. (a) FlagHisOgg1VMA-CBD Coomassie stained SDS-PAGE comparison of “On column” to “In solution ligations.” “On column” was tested with 2 mM C[K-fluorescein] (gel column F) on chitin resin (marked “Rsn” on gel) for 20 hr at room temperature. “In solution” ligations tested with Ni purified FlagHisOgg1VMA-CBD (marked P on gel) in 2 mM cysteine, 0.2 M MESNA at varying temperatures; 40 hr timepoint shown. (b) Coomassie stained SDS-PAGE of FlagHisOgg1GyrA-CBD in solution ligations, where DTT (marked D on gel) was used to generate FlagHisOgg1 with no C-term label for 20 hours at room temperature. “In solution” ligations were done in the presence of 2 mM Cys-Lys dipeptides and 0.2 M MESNA at room temperature overnight. (c) LC/MS confirmation of successful ligation and non-labeled control. (d) Catalytic activity of various Ogg1 constructs was assayed.
