Production of CuMVTT Virus-Like Particles

The production of CuMV_TT-VLP was described in detail in Zeltins et al. [28 (link)]. Briefly, Escherichia coli C2566 cells (New England Biolabs, Ipswich, MA, USA) were transformed with the CuMV_TT coat protein (CP) gene-containing plasmid pET CuMV_TT. The expression was induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The resulting biomass was collected by low-speed centrifugation and was frozen at −20 °C. After thawing on ice, the cells were suspended in the buffer containing 50 mM sodium citrate, 5 mM sodium borate, 5 mM EDTA, and 5 mM 2-mercaptoethanol (pH 9.0, buffer A) and were disrupted by ultrasonic treatment. Insoluble proteins and cell debris were removed by centrifugation (13,000 rpm, 30 min at 5 °C, JA-30.50 Ti rotor, Beckman, Palo Alto, CA, USA). The soluble CuMV_TT CP protein in clarified lysate was pelleted using saturated ammonium sulfate (1:1, vol/vol) overnight at 4 °C. Soluble CuMV_TT CP-containing protein solution was separated from the cellular proteins by ultracentrifugation in a sucrose gradient (20–60% sucrose; ultracentrifugation at 25,000 rpm for 6 h at 5 °C (SW28 rotor, Beckman)). After dialysis of CuMV_TT-containing gradient fractions, VLPs were concentrated using ultracentrifuge (TLA100.3 rotor, Beckman, at 72,000 rpm for 1 h, +5 °C) or by ultrafiltration using Amicon Ultra 15 (100 kDa; Merck Millipore, Cork, Ireland).