Strep-Tactin® Superflow® for intact virus like particle (VLP) 4.8 MDa purification
1 liter culture was induced at an OD600 of 0.6 using anhydrotetracycline (AHT) and protein expression was performed at 37°C for 3 hours at 200rpm. Cells were then pelleted, resuspended in 20 ml Buffer W (100 mM Tris-Cl, 150 mM NaCl, 1mM EDTA) and sonicated. The insoluble material was pelleted and the crude lysate was loaded on a Strep-Tactin® Superflow® column at a flow rate of 1ml/minute. After washing off the host proteins, the VLPs fused to Strep-tag® II were eluted using 2.5mM desthiobiotin in Buffer W. pASK-IBA7 was used for cloning.
For the purification of intact VLPs, the careful choice of both an ideal affinity tag system and the resin support is important:
- The affinity chromatography process should be gentle in order to keep the VLPs intact, as with Strep-tag®.
- The resin support packing within the column must allow penetration of the large VLPs.
While MacroPrep® carriers did not meet criterion (2), since the viral particles accumulated on top of the column, Sepharose® and Superflow® enabled purification. Superflow® can be used in FPLC systems, whereas Sepharose® is only suitable for gravity flow purification.