Editing VLP Expression and Purification (section)

== Expression and Purification ==

[https://jvi.asm.org/content/76/11/5784.full https://jvi.asm.org/content/76/11/5784.full]

E. coli JM105 transformed with pBL410 was grown in Trypticase yeast extract medium containing 100 μg of ampicillin per ml. At an A600 of 0.7, IPTG was added to a final concentration of 5 mM, and the bacterial suspension was further incubated at 37°C for 2 h. Cells were collected by centrifugation, and the pellets were frozen in liquid nitrogen. After thawing on ice, the cells were resuspended in 10 mM Tris-HCl (pH 8.0)-100 mM NaCl-1 mM EDTA, disrupted by lysozyme treatment (25 mg/ml for 30 min at room temperature), and sonicated (five bursts, 30 s and 80 W each, on ice). Cellular debris was removed by centrifugation at 10,000 rpm (SS34 rotor). Proteins were precipitated from the supernatant with 60% (wt/vol) ammonium sulfate and collected by centrifugation. The ammonium sulfate pellet was resuspended in 100 mM NaCl-10 mM Tris-HCl (pH 7.5)-0.1 mM MgSO4-0.01 mM EDTA. The suspension was then applied to a 1.6- by 40-cm column of Sepharose CL-4B (exclusion limit, 2 × 107 Da; Pharmacia-LKB, Freiburg, Germany) and eluted in the same buffer. The position of the elution of whole authentic Qβ virus had been previously determined for comparison purposes. Fractions of 5 ml each were collected. Aliquots of proteins present in each fraction were separated by denaturing polyacrylamide gel electrophoresis (28). Coat protein was visualized by Western blotting and Coomassie brilliant blue staining. Peak fraction 6 appeared to contain highly pure coat protein and was therefore chosen for further concentration with Centricon-30 columns (Amicon, Witten, Germany).