Recombinant GST-HINT, GST-LysRS, GST-cGAS, GST-STING (amino acids 139 to 378), and GST-ZfLysRS were produced in Escherichia coli BL21 cells. E. coli transformed with GST or GST expression constructs were grown in LB medium at 37°C to an absorbance at 600 nm of 0.6 before induction with 0.25 mM isopropyl-β-d-1-thiogalactopytanoside overnight at 16°C. Bacteria were harvested by centrifugation, resuspended in TETN-100 buffer [50 mM tris-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100, supplemented with lysozyme (2 mg/ml; Sigma), 10 mM β-mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)], and incubated on ice for 30 min. Salt and detergent concentrations were increased, respectively, to 400 mM and 0.5% before sonication. Lysates were resuspended in TETN-400 buffer [50 mM tris-HCl (pH 8), 400 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100, supplemented with 10 mM β-mercaptoethanol and 0.5 mM PMSF] and were clarified by centrifugation at 13,000 rpm for 30 min at 4°C before incubation with the appropriate volume of glutathione-Sepharose beads for 4 hours at 4°C. Sepharose beads were washed three times with ice-cold TETN-400, and recombinant proteins were eluted with elution buffer [150 mM NaCl and 50 mM tris-HCl (pH 8)] supplemented with 30 mM reduced l-glutathione. Eluates were quantified by Coomassie staining.

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