Editing QBeta VLP

132 aa coat protein of phage Qβ recombinantly expressed in E. coli
(i.e. no replicating phage RNA, infectious phage particles etc. around)
• Expression host: E. coli RB791 (K-12 derivative)
• Expression: inducible (Ptac)
• Optimized S/D sequence
• Two stop codons in tandem (no read-through)
• Plasmid-encoded resistance: kanR
• Spontaneous assembly of Qbeta particles
within the E. coli cytoplasmic space

E. coli BL21 (DE3) (Invitrogen) cells harboring the appropriate plasmids were grown in either SOB (Difco) or MEM11 supplemented with carbenicilin and spectinomycin at 50 and 100 μg/mL, respectively. Starter cultures were grown overnight at 37 °C, and were used to inoculate larger cultures. Induction was performed with 1 mM IPTG at an OD600 of 1.0 in SOB or 2.0 in MEM for 4 hours at 37 °C for all XFP constructs. Cells were harvested by centrifugation in a JA-17 rotor at 13,700 g (10,000 rpm) and were either processed immediately or stored as a pellet at −80 °C. The cell lysate was prepared by resuspending the cell pellet with 5 mL Qβ buffer (20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2) or TBS and sonicating at 30W for 3 min with 5-second bursts and 5-second intervals. Cell debris was pelleted in a JA-17 rotor at 27000 g (14,000 rpm) and 2M ammonium sulfate was added to the supernatant to precipitate the VLPs. These were pelleted and resuspended in 0.5 mL of Qβ buffer or TBS. Lipids and membrane proteins were then extracted from particles with 1:1 n-butanol:chloroform; VLPs remain in the aqueous layer. Crude VLPs were further purified by sucrose density ultracentrifugation (10–40% w:v). Particles were either precipitated from the sucrose solution with 10% w:v PEG8000 or pelleted out by ultracentrifugation in a 70.1 Ti rotor (Beckman) at 450,000 g (70,000 rpm) for at least 2 hours. After assessment of purity, additional sucrose gradients were used to further purify VLPs to >95% if necessary.