Coding sequences of virus-like particles (VLPs, 4.8 MDa) were cloned into pASK-IBA7 to obtain proteins localized to the cytoplasm and N-terminally tagged with Strep-tag®II. The plasmid was transformed into E. coli and the cell culture (1 l) was induced at OD600 of 0.6 by addition of anhydrotetracycline (AHT). Protein expression was performed at 37 °C for 3 h at 200 rpm. Afterwards, cells were harvested, resuspended with 20 ml 1x Buffer W (100 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA), and sonicated. The insoluble material was pelleted, and the lysate was applied to a Strep-Tactin® Superflow® cartridge (1 ml/min flow rate). After washing, the target protein was eluted with 2.5 mM desthiobiotin. Purification results were analyzed by SDS-PAGE. Marker (M) and two different elution fractions of VLPs fused to Strep-tag®II (lane 1 and 2) are shown.
Data kindly provided by L. Stöckl and B. Brandenburg, Robert-Koch-Institute, Berlin. Brandenburg, et al. (2005): A Novel System for Efficient Gene Transfer Into Primary Human Hepatocytes Via Cell-Permeable Hepatitis B Virus–like Particle, HEPATOLOGY (42), 1300-1309.