Figure 2

Monomeric caspase-4(C258A) and CARD purified from E. coli (a) A 4–20% SDS-PAGE gel comparing the expression levels of recombinant proteins in two different E. coli hosts. Whole cell lysates from pET28b empty vector (Lane 1), pET28b harboring the CARD domain of caspase-4 (Lane 2), and pET28b harboring caspase-4 (C258A) (Lane 3). The amount of protein loaded on the SDS gel was normalized to a cell culture biomass based on OD₆₀₀. SEC elution profiles of caspase-4 (C258A) (b) and CARD domain (c) from Superdex 200 10/300 gl columns calibrated with protein standards and SDS-PAGE gels of the purified caspase-4 (C258A) (b) and CARD domain (c). 5 µg of purified protein was loaded in each lane. Calibration curves are presented in insets and enlarged calibration curves are presented in Supplementary Fig. S5. (d) Molecular weight (MW) determination of purified caspase-4 (C258A) and CARD domain from ClearColi BL21(DE3) by analytical ultracentrifugation (AUC) and theoretical MW. Shown are the mean values ± standard deviation. SDS-PAGE gels shown in a, b, and c were cropped for clarity and full-length gels are presented in Supplementary Figs S1 and S4.