Purification of Recombinant GSDME and CASP

Recombinant GSDMEs and CASPs are all soluble and were purified as described previously (Jiang et al., 2020 (link); Xu et al., 2022 (link)). Briefly, the CDSs of TrGSDME and CASP variants were each cloned into pET30a (+), and the CDSs of HsGSDME, MmGSDME, and chimeric GSDME were each cloned into pET28a-SUMO. The recombinant plasmids were introduced into Escherichia coli Transetta (DE3) (TransGen, Beijing, China) by transformation. The transformants were cultured in Luria broth (LB) at 37°C until logarithmic growth phase. Isopropyl-β-d-thiogalactopyranoside (0.3 mM) was added to the medium, followed by incubation at 16°C for 20 hr. Bacteria were harvested and lysed, and the supernatant was collected for protein purification with Ni-NTA columns (GE Healthcare, Uppsala, Sweden). The proteins were dialyzed with PBS at 4°C and concentrated.