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From 2012.igem.org

Purification of VLPs

After production, our sample still contains differnt types of contaminants. To remove the contaminants we build further upon the basic purification protocol that was given to us by Richard Kormelink. We changed the ultracentrifuge and the FPLC for a even more purer sample.

(PICTURE OF ULTRACENTRIFUGE OVERVIEW)

The purification process consists of three parts. The first part is differential centrifugation. It is ultracentrifugation with a sucrose gradient, this technique relies on that smaller particles will get less far through the sucrose solution than larger particles. The result will look like a smear on the side of the vial. We take the lowest part of the smear, in which our VLPs contain. Most of the contaminants in the sample is now removed, to make it purer we load the sample onto a FPLC column. FPLC or Fast Protein Liquid Chromatography is a form of liquid chromatography that is often used for purifying mixtures of proteins. The FPLC can separate proteins on particle size, charge distribution(ion-exchange), hydrophobicity or reverse-phase. We used a column that separate the particles on particle size, these kind of columns do not interact with proteins, so that are particles remain intact. The output of the FPLC gives us multiple fractions of a few mL. In these fraction our VLPs, aggregates or other proteins are residing. This can be checked with the DLS or if you have calibrated the column it is possible to predict in which fraction the VLPs are.

(PICTURE OF FPLC OVERVIEW)

The complete purification methods has so far only being used with the wild type CCMV VLPs in our lab. Multiple variations of CCMV and HepB has been purified only by ultracentrifuge and was in the lab successful.