Cellular pellets from bacteria transformed with the different N TAIL expression plasmids were resuspended in 5 volumes (v/ w) buffer A (50 mM sodium phosphate at pH 8, 300 mM NaCl, 10 mM Imidazole, 1 mM phenyl-methyl-sulphonyl-fluoride [PMSF]) supplemented with lysozyme 0.1 mg/mL, DNase I 10 mg/mL, protease inhibitor cocktail (Sigma) (50 mL/g cells). After a 20-min incubation with gentle agitation, the cells were disrupted by sonication (using a 750 W sonicator and four cycles of 30 sec each at 60% power output). The lysate was clarified by centrifugation at 30,000g for 30 min. Starting from a 1-L culture, the clarified supernatant was incubated for 1 h with gentle shaking with 4 mL Chelating Sepharose Fast Flow Resin preloaded with Ni 2+ ions (Amersham Pharmacia Biotech), previously equilibrated in buffer A. The resin was washed with buffer A, and the N TAIL proteins were eluted in buffer A containing 250 mM imidazole. Eluates were analyzed by SDS-PAGE for the presence of the desired product. The fractions containing the recombinant product were combined, and concentrated using Centricon Plus-20 (molecular cutoff, 5000 Da) (Millipore). The proteins were then loaded onto a Superdex 75 HR 10/30 column (Amersham Pharmacia Biotech) and eluted in either 10 mM sodium phosphate at pH 7 or 10 mM Tris/HCl at pH 8. The proteins were stored at À20 C.