Purification and Characterization of CASP4-nleF Complex

BL21 Star cells were co-transformed with pACYC-DUET-1-CASP4^C258S His-p20/p10 and pMAL-c2x-nleF_EPEC. Bacterial pellets were re-suspended in 20mM Tris-HCl pH 7.4, 250 mM NaCl and lysed by sonication and purified by amylose affinity chromatography. Bacterial lysates were incubated with amylose resin for 1.5 h at 4 °C and then washed with 50 ml wash buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl and eluted with wash buffer supplemented with 10 mM maltose. The co-elute was dialysed and then purified further by IMAC talon affinity chromatography, as described previously(41 (link)). Complex formation was analysed by size exclusion (Akta prime) with a Superdex200 column (GE Healthcare) using the Gel Filtration Markers Kit for Protein Molecular Weights 12,000-200,000 Da (Sigma-Aldrich) to determine complex size. Size exclusion fractions were verified by SDS PAGE gel and confirmed by Mass spectrometry.