Fig. 5. Condensation domain assay for the final module of the teicoplanin NRPS. Initially, peptidyl-CoA substrates prepared by solid phase peptide synthesis are loaded onto the isolated PCP domain from the preceding module using the promiscuous phosphopantetheinyl transferase Sfp (top left), after which these substrates are then added to the Tcp12_ΔTE2 construct along with Dpg and ATP in order to assess the formation of Dpg-extended peptide products. Products peptides are extended by the addition of Dpg through the actions of Tcp12_ΔTE2 (green box, residual starting peptide shown in the red box). All peptides can either remain PCP-bound at the end of the assay (where they are then analysed as their methylamides through the addition of methylamine) or they can be hydrolysed from the PCP. Peptide structures synthesised and used as substrates in these assays are shown in the boxed area on the right of the figure (1–9, D-1, D-4). NRPS domain descriptions: C, condensation; A, adenylation; PCP, peptidyl carrier protein; X, P450 (Oxy) recruitment.
