High-capacity purification of His-tagged proteins by affinity membranes containing functionalized polymer brushes.

Porous membrane absorbers are attractive for increasing the rate of protein purification, but their binding capacity is low relative to porous beads. Modification of membranes with functionalized polymer brushes, however, can greatly enhance capacity. This work demonstrates that membrane modification with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes derivatized with nitrilotriacetate-Ni2+ (NTA-Ni2+) complexes allows purification of polyhistidine-tagged ubiquitin (HisU) in less than 30 min with a binding capacity of 120 mg of HisU/cm3 of porous alumina membrane. Adsorption isotherms show that saturation of the brushes occurs at HisU concentrations as low as 0.04 mg/mL and that these brushes can bind up to 23 monolayers of HisU. Gel electrophoresis reveals that the purity of eluted HisU is more than 99%, even when the initial feed solution contains 10% bovine serum or a 20-fold excess of BSA. Thus, reusable porous membranes modified by PHEMA-NTA-Ni2+ brushes are attractive candidates for rapid purification of polyhistidine-tagged proteins.