Utilization of the Polymer Sieving Effect for the Removal of the Small Molecule Biotin-CDM

After labeling of proteins with chemical tags, the unconjugated tag that remains in solution must be removed to prevent interference with downstream workflows. Currently, unconjugated tags are removed using dialysis or spin filtration, which suffers from significant sample loss due to protein precipitation, transfer steps, and lengthy protocols. In this study, polymer-based protein engineering was used to sequester the small molecule biotin. By enclosing avidin in a polymer cage, known as caged-avidin, free, unconjugated biotin was able to bind to avidin’s active site, while more than 90% of biotinylated proteins were excluded. In the presence of excess biotin, less than 10% of biotinylated protein bound to avidin-coated beads (known as NeutrAvidin) due to competition from the free biotin. However, sequestration of the excess free biotin by caged-avidin allowed recovery of up to 93% binding of biotinylated proteins to NeutrAvidin beads. Utilization of caged-avidin enabled the use of biotin-CDM, a reversible biotin tag, for tagging, capture, cleanup, and release of a single protein sample and a whole yeast proteome, improving the final protein yields to 97%. This study demonstrates an application for the sieving effect of polymers on the surface of proteins to sequester and remove small molecules from solution without dialysis.