Figure 6.

Expression and purification of recombinant darcin (r-darcin). E.coli BL21(λ)DE3 cells wer e transformed with a pET28b plasmid and at hourly intervals after induction cells were removed and lysed in water. The equivalent of 0.1 A600 of lysate was loaded into each lane of a 15%(w/v) polyacrylamide gel (panel A). The cell lysate was passed through a 1.2 mm filter and applied directly to a NiNTA metal affinity column column equilibrated with 50 mM sodium phosphate, 10 mM imidazole, 0.3 M NaCl pH8.0. The column was washed in the same buffer containing 20 mM imidazole and bound r-darcin was eluted by increasing the imidazole concentration to 250 mM (panel B). The purified r-darcin was subjected to in gel digestion with endopeptidase LysC and the peptides were analysed by MALDI-ToF mass spectrometry. All LysC peptides of mass greater than 800Da were readily detected in the MALDI-ToF spectrum, confirming the expression of the correct protein (panel C).