BIOT 442

The mechanism of purification requires three sequential steps: I - The target is specifically precipitated in the presence of a non-immobilized modified ligand whereas impurities are left soluble in the supernatant and are removed by centrifugation. II - The resulting pellet is then washed once with minute volume of buffer to remove traces of impurities. III - The target is eluted from the pellet while keeping the modified ligand insoluble in the precipitate. The approach was implemented in two distinct platforms each exploiting a different complexing chemistry, either the: [Desthiobiotin : Avidin] complex or the [Catechol : Fe3+] complex and has been demonstrated with four distinct ligands (Protein A, Protein G, Concanavalin A and Fluorescein) leading to relatively high yields (75-94%) and purity (88-97%) within 20-25 minutes overall time. The purity obtained was shown not to be affected by substantial increase in the contamination background.

In addition, greater purity and yield, as well as a significantly faster isolation process were observed when free rather than immobilized Protein A was used in a comparative study. This provided direct evidence of the advantages derived from binding under homogenous solutions together with the exclusion of polymeric resins from the process. Preliminary results and possible implementation of the approach for: Fusion proteins (e.g. His-tag proteins, GST-proteins); Membrane proteins (e.g. Na,K-ATPase); Eukaryotic mRNA and Cells will be presented and discussed.