Figure 1.

Purification and ATPase activity of MBP-NBD variants. (A) E. coli strain BL21Gold cells expressing the various constructs were disrupted in column buffer (20 mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA) by sonication and the lysate clarified by centrifugation. MBP fusion proteins were purified by amylose affinity chromatography and eluted in 20 mM Tris pH 7.4, 200 mM NaCl, 10 mM Maltose. 1 µg of each purified protein was separated by SDS-PAGE and stained with Coomassie Blue R250. NBD1 comprises amino acids 442 to 685 and NBD2 comprises amino acids 1091 to 1337 delimited by disordered regions present in CTS modeled on homology with Sav1866 (2HYD.pdb) as a structure template.¹ (B) Purified proteins were incubated in 50 mM Tris pH 7.4, 0.15 mM NH₄Cl, 5 mM MgSO₄ in a 10 µl final volume. Reaction was started by the addition of ATP and incubated at 37°C for 30 min. Phosphate release was measured by the method of Chifflet et al. 1988.¹⁸,¹⁹ Data are means ± SD of the mean and are representative of 3 independent experiments. (C) Equivalent amounts of purified proteins were loaded on a SDS gel and Coomassie stained (left) or transferred onto a nitrocellulose membrane and blotted with an anti-GroEL antibody (right).