Editing VLP Expression and Purification (section)

== AP205 ==

Plasmids were transformed into E.coli JM109. A seed culture<br/>was prepared by inoculating a single colony into LB medium<br/>containing 20 mg/l Ampicillin and growing the culture overnight<br/>at 37uC without shaking. For expression, the overnight culture was<br/>diluted in M9 medium supplemented with casaminoacids (Difco)<br/>and containing 20 mg/l Ampicillin and growth of the culture<br/>carried out at 37uC with vigorous aeration for 14–20 hours.<br/>Cells were resuspended in TEN buffer (20 mM Tris-HCl, 5mM<br/>EDTA, 150 mM NaCl, pH 7.8) containing 1 mg/ml lysozyme<br/>and 0.1% Tween 20, and lysed by three freeze thaw cycles<br/>followed by ultrasonication. Clarified lysates were purified by one<br/>or more sequential gel filtration steps using CL-4B, CL-2B and<br/>Sepharose 6B columns (GE Healthcare). Some VLPs were<br/>additionally purified by ultracentrifugation over a CsCl gradient.<br/>Concentrations of the purified proteins were determined by the<br/>Bradford test. For the AP205-GnRH vaccine, two bands reactive<br/>with an anti-GnRH antiserum were observed when using vector<br/>pAP405 for expression. This wasn’t observed in any other<br/>construct. Subcloning into a pKK222 derivative however led to<br/>a single band. The immunogenicity of both constructs was<br/>equivalent.<br/>Electron


[[Category:VLP]]