cGAS Full-Length and Catalytic Domain Purification

The cDNA of cGAS full length and catalytic domain were cloned into a modified pET-28a vector with an N-terminal Avi-His₆-SUMO tag. Mouse cGAS catalytic domain (residues 142 – 507) was expressed in E.coli BL21 (DE3) with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) induction overnight at 16°C and purified as described previously^{19 (link)} . Biotin-Avi-His₆-SUMO human and mouse cGAS full length and catalytic domains (human cGAS domain residues 157 – 522) were expressed in E.coli BL21 (DE3) cells co-transformed with the plasmids coding for cGAS and the pBirAcm plasmid coding for BirA. Protein expression was induced with 0.4 mM IPTG in the presence of 5 μg/ml biotin (Sigma-Aldrich, B4501). The proteins were first purified using a Ni²⁺-NTA column (Qiagen) and were further purified over a Superdex200 column (GE Healthcare Life Sciences). Biotin-Avi-His₆-SUMO human and mouse cGAS full length and human cGAS catalytic domain were eluted with the buffer containing 20 mM Tris, 500 mM NaCl, pH 7.5. Biotin-Avi-His₆-SUMO mouse cGAS catalytic domain was eluted with the buffer containing 20 mM Tris, 150 mM NaCl, pH 7.5. All mutants were generated using a PCR-based technique with appropriate primers and confirmed by DNA sequencing. The cGAS mutant proteins were expressed and purified the same way as the wild-type cGAS.