Escherichia coli BL21(DE3) cells harbouring the expression vectors were grown overnight in 50 ml LB broth containing ampicillin or kanamycin at 37°C. A 1:100 dilution of the overnight culture was used to inoculate 2 L fresh LB broth to an OD600 of 0.6 and then cooled to 16°C. IPTG was added to a final concentration of 1 mM, and incubations were continued at 16°C for 10 h. Cells were harvested by centrifugation at 6000 g for 10 min and resuspended in a lysis buffer containing 20 mM Tris-HCl (pH 8.0), 50 mM NaCl and 5% glycerol. Lysis was completed by sonication on ice, the soluble fractions were collected by centrifugation, and then loaded onto a Ni-NTA affinity column (Qiagen). The proteins were eluted with an increasing concentration gradient of imidazole and then purified by gel filtration chromatography. Peak elution fractions were analysed by electrophoresis on 10% or 12% SDS-PAGE gels. Fractions containing pure proteins were pooled, concentrated in an Amicon apparatus (Millipore) with a 10 kDa molecular weight cut-off membrane, and then stored in 0.1 ml aliquots at -80°C. The concentrations of proteins were determined with a BCA protein assay kit (Pierce) according to the manufacturer's protocol.