All genes were cloned into E. coli cells (BL21 Lemo21 (DE3)) for expression, using auto-induction41 at 18° or 37 °C for 16–24 h in 500 mL scale. Post-induction, cultures were centrifuged at 8000 × g for 15 min. Cell pellets were then resuspended in 25–30 mL lysis buffer (TBS, 25 mM Tris, 300 mM NaCl, pH8.0, 30 mM imidazole, 0.25 mg/mL DNase I) and sonicated for 2 min total on time at 100% power (10 s on/off) (QSonica). Lysate was then centrifuged at 14,000 × g for 30 min. Clarified lysates were filtered with a 0.7 μm syringe filter and put over 1–4 mL of Ni-NTA resin (QIAgen), washed with wash buffer (TBS, 25 mM Tris, 300 mM NaCl, pH8.0, 60 mM imidazole), then eluted with elution buffer (TBS, 25 mM Tris, 300 mM NaCl, pH8.0, 300 mM imidazole). Eluate was then concentrated with a 10,000 m/w cutoff spin concentrator (Millipore) to ~0.5 mL based on yield for SEC.

D2 proteins went through an extra round of bulk purification. Concentrated protein was heated at 90 °C for 30 min to further separate bacterial contaminants. Samples were then allowed to cool down to room temperature and any denatured contaminants were removed by centrifuging at 20,000 × g.

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