Gel filtration chromatography

FGFRs were prepared as previously described^{6 (link),34 (link),44 (link),45 (link)}. Briefly, FGFR1 (residues 458–765), FGFR2 (residues 458–768), FGFR3 (residues 450–758) as well as FGFR4 (residues 445–753), and their mutants, FGFR1^C584S, FGFR1^V561M, FGFR2^V564I, FGFR2^V564F and FGFR3^V555M, were cloned into a modified pET28a vector in frame with an N-terminal PreScission-cleavable 6×His tag and expressed in E. coli BL21 Rosetta cells. For crystallization, FGFR1^C584S was co-expressed with untagged YOPH to induce non-phosphorylated proteins. The harvested cell pellets were lysed in a buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole and 0.5 mM TCEP, and FGFRs were purified over Ni-NTA resin followed by enzymatic digestion with PreScission for 6×His-tag cleavage and further purified by anion exchange chromatography (GE Healthcare). For crystallization, FGFR1^C584S was further purified by gel filtration chromatography (GE Healthcare). The proteins were concentrated to 5–16 mg/mL and stored at −80 °C.

The AP205 VLP prokaryotic expression plasmid was amplified and expressed in Escherichia coli (E. coli). The procedure of VLP purification and identification followed previous study^{2 (link)}. In brief, BL21 E. coli cultured overnight were lysed completely by ultrasound. The lysate was purified by acidification, sedimentation of saturation ammonium sulfate, hydrophobic interaction chromatography (GE Healthcare), and gel filtration chromatography (GE Healthcare). Dimer was produced through depolymerization of purified VLP, sedimentation, resolvation and purification by hydrophobic interaction chromatography. N-Ethylmaleimide (NEM, Sigma Aldrich) was used to block sulfhydryl group in dimer protein to avoid useless thioether bonding with crosslinkers. Single ATR001 peptide (A-F-H-Y-E-S-Q) and FITC conjugated-ATR001 peptide were customized from GL biochem of Shanghai. Analyzed by high performance liquid chromatography and mass spectrometry, the purity of peptides reached 95%. Peptides were covalently conjugated to VLP and dimer respectively in a mass ratio of 1:3 through Sulfo-SMCC crosslinker (Pierce Biotechnology) to produce ATR-AP205-001 vaccine and ATR-Dimer-001 vaccine. Conjugation efficiency of the productive vaccines was identified by SDS-PAGE (see Supplementary Fig. S7). Coomassie Brilliant Blue protein assay kit was used to detect vaccine concentration.

RIG-I and RIG-I E373Q were expressed and purified from insect cells as described previously (Cui et al., 2008 (link)). Briefly, sequences encoding RIG-I were cloned into pFBDM vectors and transformed into E. coli DH10MultiBac cells. Bacmids were extracted for transfection into SF9 insect cells and propagated virus was used for protein expression in High Five insect cells. Seventy-two hours after infection cells were harvested and flash frozen in liquid nitrogen. RIG-I Δ2CARD was expressed in E. coli BL21 Rosetta (DE3), using pET expression vectors as described earlier (Cui et al., 2008 (link)). All recombinant proteins were purified using metal affinity (QIAGEN, Germany), heparin affinity and gel filtration chromatography (both GE Healthcare, Buckinghamshire, UK). Fractions containing RIG-I were concentrated to 6 mg/mL and flash-frozen in liquid nitrogen.