Wild type and mutant pET-p66 expression vectors were respectively transformed into E. coli BL21(DE3) and the expression was induced with 0.3 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) when E. coli grew up to an OD600 of 0.7,1.0. E. coli expressing p66 was spun down and re-suspended in the binding buffer (50 mM NaH 2 PO4, 300 mM NaCl, 10 mM imidazole). After the bacteria were disrupted by ultrasonication and centrifuged at 15 000 g for 30 min at 4uC, the wild type and mutant p66 in supernatants were purified using Ni-NTA magnetic agarose beads (Qiagen, Germany). The purified p66 proteins were subjected to NativePage Novex Bis-Tris gel analysis and Western blotting according to the protocol recommended by Invitrogen, USA.