Protein isolation and digestion are often vital steps in studies of protein structures, interactions and post-translational modifications. Porous membranes present an attractive platform for rapid proteolysis and protein purification because convective flow through pores quickly transports proteins or reagents to functional sites. This dissertation demonstrates functionalization of porous membranes with metal-chelating polyelectrolytes, peptide ligands, and enzymes, to create methods for fast protein purification, affinity tag removal and protein digestion. Additionally, attachment of functionalized membranes to pipette tips enables especially rapid and convenient protein digestion or isolation. Development of high-capacity affinity membranes for protein isolation requires membrane pores coated with thin films that bind multilayers of proteins. To prepare membranes that selectively capture polyhistidine-tagged (His-tagged) proteins, this work explores layer-by-layer adsorption of polyelectrolytes containing chelating groups that form Ni2+ complexes. Sequential adsorption of protonated poly(allylamine) (PAH) and carboxymethylated branched polyethyleneimine (CMPEI) leads to membranes that bind Ni2+ and capture ~60 mg of His-tagged ubiquitin per mL of membrane. Both binding capacity and metal-ion leaching are similar to values seen with high-binding commercial beads, but membranes should facilitate protein isolation in minutes.After purification, fusion tag removal is often an essential step prior to protein characterization. Removal of small ubiquitin-like modifier (SUMO) tags in SUMO-protease-containing membranes served as a proof-of-concept demonstration for in-membrane tag removal. The time required for tag removal is similar with dissolved and immobilized His-tagged SUMO protease, but the membrane is reusable, and immobilized proteases retain much of their activity after three uses.Membranes are also convenient substrates for trypsin immobilization and subsequent proteolysis. Passage of protein solutions through 100-μm thick trypsin-modified membranes enables reaction residence times as short as milliseconds to limit digestion and provide large peptides for mass spectrometry (MS) analysis. Large peptides can both enhance protein sequence coverage and help identify flexible regions in a protein. With either cytochrome c or apomyoglobin, in-membrane trypsinolysis cleaves the protein after lysine residues in highly flexible regions to generate two large peptides that cover the entire protein sequence. Further combining membrane techniques with pipette tips yields a convenient platform for rapid protein purification and digestion. Pushing a protein-containing solution through a trypsin-modified membrane at the end of a pipette tip digests proteins in <30 s, and enables tryptic digestion without alkylation of cysteine residues. Similarly, when membranes contain Ni2+ complexes, pipetting aqueous His-tagged protein through the membrane and subsequent rinsing and elution yield purified protein in 2 min. These applications demonstrate the potential of functional membranes for rapid proteolysis and protein isolation.
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