Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography.

Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation.

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation.

One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide.

One-step affinity purification of fusion proteins with optimal monodispersity and biological activity: application to aggregation-prone HPV E6 proteins.

Background: Bacterial expression and purification of recombinant proteins under homogeneous active form is often challenging. Fusion to highly soluble carrier proteins such as Maltose Binding Protein (MBP) often improves their folding and solubility, but self-association may still occur. For instance, HPV E6 oncoproteins, when produced as MBP-E6 fusions, are expressed as mixtures of biologically inactive oligomers and active monomers.