Modified Membranes for Protein Isolation or Controlled Proteolysis

Porous membranes are attractive for separations and catalysis because convective mass transport rapidly brings analyte molecules to binding or catalytic sites. The minimal thickness of commercial microporous membranes (~100 mm) also leads to low pressure drops, and variation of flow rate affords fine control (milliseconds) over residence times. To create functional membranes, we adsorb polyelectrolytes in membrane pores and subsequently covalently or electrostatically attach affinity molecules or enzymes to the polyelectrolytes. Immobilization of Ni²⁺-nitrilotriacetate complexes in pores yields membranes that capture polyhistidine-tagged proteins, and spin membranes (now available commercially) facilitate high-capacity purification of tagged proteins in under 5 min. The use of polyelectrolyte films leads to equilibrium binding capacities as high as 90 mg of protein per mL of membrane. Antibody mimotopes anchored in membranes capture specific therapeutic monoclonal antibodies from human serum, and we aim to use these membranes for rapid antibody quantitation. Immobilized enzymes such as trypsin and pepsin digest proteins in membrane residence times as short as a few milliseconds, and the size of the proteolytic peptides increases with increasing flow rates to facility mass spectrometry characterization of proteins.