The following CP mutants were cloned and expressed in pET28a vector (Addgene): N-terminal deletion mutants (ΔN9, ΔN19, ΔN29, ΔN39, ΔN49, ΔN59, and ΔN69), C-terminal deletion mutants (ΔC10, ΔC20, ΔC30, ΔC40, ΔC50, and ΔC60), and double deletion mutants (ΔN49C40, ΔN39C60, and ΔN29C40). Sequences were verified by nucleotide sequencing (Eurofins Genomics). Since we have shown that a hexahistidine (His)–tag at the C terminus of the wild-type CP does not affect filament formation (Fig. 5B), mutants ΔN49, ΔN69, and ΔN49C40 were prepared with C-terminal His-tags, followed by a tobacco etch virus (TEV) protease cleavage site to simplify purification in the absence of filaments. All CP mutant proteins were expressed in BL21(DE3) cells under the same conditions as described for VLPs. After expression, cells were lysed using an EmulsiFlex-C5 high-pressure homogenizer (Avestin) in PBS buffer (pH 7.4) containing 5% glycerol because sonication in some cases resulted in insoluble proteins. The presence of filaments in lysates was monitored by negative-stain TEM. CP mutant proteins ΔC40 and ΔC60 were further purified following the VLP purification procedure described above. His-tagged mutant proteins (ΔN49, ΔN69, and ΔN49C40) were purified on Ni–NTA (nitrilotriacetic acid) columns (Qiagen), and bound proteins were eluted with PBS buffer (pH 7.4) containing 5% glycerol and 300 mM imidazole. Eluted samples were dialyzed against PBS buffer (pH 7.4) containing 5% glycerol. For further purification, ΔN49 and ΔN69 were dialyzed in the presence of TEV protease to cleave the His-tag. ΔN49 and ΔN69 mutants were subjected to a second Ni-NTA chromatography with the cleaved protein eluting in the unbound fraction. ΔN49 and ΔN69 from unbound fractions or ΔN49C40 with intact C-terminal His-tag after dialysis was further purified by size exclusion chromatography using Superdex 200 10/300 GL (24 ml) or Superdex 200 16/60 PG (120 ml) columns (GE Healthcare). The running buffer for ΔN49 was 15 mM tris-HCl, 130 mM NaCl, and 5% glycerol (pH 7.4), whereas the running buffer for ΔN69 and ΔN49C40 was PBS and 5% glycerol (pH 7.4). After chromatography, fractions containing pure proteins were pooled and concentrated in Amicon Ultra centrifugal filters (10,000 or 30,000 molecular weight cutoff) (Millipore). The final solutions of ΔN69, ΔC40, and ΔC60 with their respective concentrations of 0.9, 0.7 to 0.8, and 0.8 to 1.0 mg/ml were stored at 4°C. ΔN49 and ΔN49C40 were concentrated to 10 mg/ml and stored at −80°C.

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