Heparin column

HEK293T Riplet^−/− cells were collected and resuspended in Buffer A. The cells were broken by electronic homogenizer and centrifuged at 100,000 g for 30 min to get S100. S100 fraction was loaded onto Mono-Q sepharose column (GE healthcare) preequilibrated with Buffer C (Hepes 20 mM pH 7.5, DTT 1 mM and PMSF 1 mM) and the flow-through fraction was collected as Fraction A. The column was then washed with Buffer D (Hepes 20 mM pH 7.5, NaCl 0.5M, DTT 1 mM and PMSF 1 mM) and Fraction B was collected. Fraction C was eluted with Buffer E (Hepes pH 7.5 20 mM, NaCl 1M, DTT 1 mM and PMSF1 mM). Fraction A was loaded onto Heparin column (GE healthcare) preequilibrated with Buffer C, and active fractions were eluted with buffer Buffer E, which were pooled and subjected to buffer-exchange to Buffer F (NaAc 20 mM pH 6.0, DTT 1 mM and PMSF 1 mM). Active fractions in Buffer F were loaded onto mini-S sepharose column (GE healthcare). Active fractions from mini-S column were eluted with Buffer G (NaAc 20 mM pH 6.0, NaCl 1 M, DTT 1 mM and PMSF 1 mM), which were loaded onto a Superdex200 column (GE healthcare) preequilibrated with Buffer H (Hepes 20 mM pH 7.5, NaCl 150 mM, DTT 1 mM and PMSF 1 mM). Fractions from Superdex200 column were subjected to SDS–PAGE and the cell-free assay to examine RIG-I and MAVS activation. All procedures were carried out at 4 °C by using ÄKTA or ÄKTAmicro system.

The coding gene of FGF2 (residues 143–288) was synthesized at the NdeI and XhoI sites of the expression vector pET17b (+) (Novagen). The resulting construct was transformed into Rosetta (DE3) pLysS. The transformant was grown to an OD₆₀₀ of 0.6–0.7 in LB media containing 50 μg/mL ampicillin and chloramphenicol at 37 °C, and 0.5 mM IPTG was added. After 20 h of incubation at 20 °C, cells were harvested using centrifugation, resuspended in a buffer (10 mM Tris pH 8.0 and 200 mM NaCl), and disrupted using sonication. To remove cellular debris, centrifugation was performed at 10,000×g for 40 min at 4 °C. The soluble fractions were loaded into a heparin column (GE Healthcare) and eluted using a linear gradient of 0.5–2.0 M NaCl in 10 mM Tris pH 8.0 buffer. The fractions containing target proteins were collected and finally loaded onto a HiLoad® 16/600 Superdex® 75 pg column (GE Healthcare) equilibrated with a buffer consisting of 10 mM NaH₂PO₄ (pH 8.0) and 200 mM NaCl. Purification was performed using the AKTA-FPLC system (GE Healthcare).

The LbuCas13a expression and purification were performed as our previous work^{33 (link)}. Briefly, The E. coli Rosetta (DE3) was transformed with pET-Sumo-LbuCas13a expression plasmid and grown overnight in Terrific Broth (TB) medium at 37 °C and 150 rpm until the exponential growth phase. Afterwards, protein expression was induced with 100 µM isopropyl-1-thio-b-D-galactopyranoside (IPTG) and cultured at 16 °C for 12 h. Cells were harvested by centrifugation at 5000 rpm and lysed by sonication in the lysis buffer (20 mM Tris–HCl, 1 M NaCl, 20 mM imidazole, 10% glycerol, pH 7.5). Lysate was separated by centrifugation and the supernatant was incubated with Ni-NTA agarose, the bound protein was eluted by elution buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 250 mM imidazole). The His₆-Sumo tag of LbuCas13a protein was digested by Ulp1 protease and further purified by heparin column (GE Healthcare). The purified product was dissolved in storage buffer (20 mM Tris–HCl, pH 7.5, 1 M NaCl, 50% glycerol) and stored at −80 °C until use.

Details of the DNA constructs are given in Supplementary Table S1. His6-tagged recombinant proteins were produced in BL21 (DE3) grown in MagicMedia (Invitrogen) 24 h, 28 °C and purified on HisTrap columns (GE Healthcare) by Imidazole gradient elution on AKTA Prime according to manufacturer instructions. Following tag removal by incubation with PreScisson protease (6 h, 4 °C), the protein was purified on Heparin column (GE Healthcare), eluted by NaCl gradient and dialyzed for 2 days (20 mM phosphate buffer, 100 mM NaCl, pH 7.5). For protein FITC labeling, 100 μM of dialyzed purified protein was incubated with a two-fold molar excess of fluorescein isothiocyanate in carbonate buffer (50 mM pH 9.5, 100 mM NaCl) overnight at 4 °C and free FITC was removed by dialysis (48 h, 4 °C). The efficacy of FITC incorporation was determined by SDS–PAGE and spectral analysis. The molecular FITC:protein ratio was confined to a range between 1.5 and 2 for all proteins. Gel-shift assays were performed as in ref. ^{39 (link)}. Redox-insensitive En2 covalent dimerization was obtained with the homobifunctional crosslinker 1,8-bis(maleimido)diethylene glycol (Thermo #22336), according to manufacturer instructions.