Fig. 3

ALACAT synthesis by E. coli cell-free system. Experimental design for cell-free synthesis (a). Ten small ALACATs (belonging to two series; ‘B’ and ‘H’) and one long ALACAT were synthesized in a small-scale cell-free synthesis system using a microdialysis device (b). Lysine and arginine residues of the synthesized ALACATs were labelled with ¹³C/¹⁵N. b Representative SDS-PAGE separation images of the synthesized ALACATs. His-tag purified ALACATs were separated by NuPAGE 4–12% gradient gels and visualised by CBB staining. Asterisk: ALACAT band. c Selective reaction monitoring (SRM) analysis of ALACAT peptides. The efficiency of stable isotope incorporation was estimated by SRM for two tryptic digested peptides derived from one ALACAT