Enterokinase

The pET32a plasmid containing the mouse α-syntrophin PDZ domain sequence as a thioredoxin (TRX) fusion protein, with a centrally located His-tag, was expressed and purified as in (Seedorff et al. 2010) . Briefly, the plasmid was transformed into BL21 (DE3) cells, which were grown to OD 0.8 in LB at 37°C, before induction with 0.1 mM IPTG and transferred to 30°C for 3.5 h. The culture was centrifuged and the cell pellet lysed by repetitive freezethawing, before further centrifugation. The clarified lysate was applied to a Ni-NTA column (BioRad) washed, and eluted with increasing concentrations of imidazole. Eluted fractions were combined and buffer exchanged using a PD10 desalting column (GE Healthcare) to remove imidazole, before treatment with enterokinase (Genscript, 50 units/10mg purified protein) at 20°C for 20 h. The His-tagged TRX domain and enterokinase were removed by reapplication to the Ni-NTA column. Purified PDZ domain was concentrated and lyophilized for storage. Immediately prior to the electrochemical experiments, the domain was resuspended and exchanged into 50 mM HEPES (pH=8.5) and 150 mM NaCl, at a final concentration of 230 μM, based on an extinction coefficient of 2980 M -1 cm -1 (λ=280 nm).