Immobilization of Carboxymethylated Polyethylenimine–Metal-Ion Complexes in Porous Membranes to Selectively Capture His-Tagged Protein

Membrane adsorbers rapidly capture tagged proteins because flow through membrane pores efficiently conveys proteins to binding sites. Effective adsorbers, however, require membrane pores coated with thin films that bind multilayers of proteins. This work employs adsorption of polyelectrolytes that chelate metal ions to create functionalized membranes that selectively capture polyhistidine-tagged (His-tagged) proteins with binding capacities equal to those of high-binding commercial beads. Adsorption of functional polyelectrolytes is simpler than previous membrane-modification strategies such as growth of polymer brushes or derivatization of adsorbed layers with chelating moieties. Sequential adsorption of protonated poly­(allylamine) (PAH) and carboxymethylated branched polyethylenimine (CMPEI) leads to membranes that bind Ni²⁺ and capture ∼60 mg of His-tagged ubiquitin per mL of membrane. Moreover, these membranes enable isolation of His-tagged protein from cell lysates in <15 min. The backbone amine groups in CMPEI likely increase swelling in water to double protein binding compared to films composed of PAH and the chelating polymer poly­[(<i>N</i>,<i>N</i>-dicarboxymethyl)­allylamine] (PDCMAA), which has a hydrocarbon backbone. Metal leaching from PAH/CMPEI- and PAH/PDCMAA-modified membranes is similar to that from GE Hitrap FF columns. Eluates with 0.5 M imidazole contain <10 ppm of Ni²⁺

Increased Protein Sorption in Poly(acrylic acid)-Containing Films through Incorporation of Comb-Like Polymers and Film Adsorption at Low pH and High Ionic Strength

In principle, incorporation of comb-like block copolymers in multilayer polyelectrolyte films can both increase film thickness relative to coatings containing linear polymers and provide more swollen films for increased sorption of proteins. In the absence of added salt, alternating adsorption of 5 bilayers of protonated poly­(allylamine) (PAH) and comb-like poly­(2-hydroxyethyl methacrylate)-<i>graft</i>-poly­(acrylic acid) (PHEMA-<i>g</i>-PAA) leads to ∼2-fold thicker coatings than adsorption of PAH and linear PAA, and the difference in the thicknesses of the two coatings increases with the number of bilayers. Moreover, the (PAH/PHEMA-<i>g</i>-PAA)_<i>n</i> films sorb 2- to 4-fold more protein than corresponding films prepared with linear PAA, and coatings deposited at pH 3.0 sorb more protein than coatings adsorbed at pH 5.0, 7.0, or 9.0. In fact changes in deposition pH and addition of 0.5 M NaCl to polyelectrolyte adsorption solutions alter protein sorption more dramatically than variations in the constituent polymer architecture. When deposited from 0.5 M NaCl at pH 3.0, both (PAH/PHEMA-<i>g</i>-PAA)₅ and (PAH/PAA)₅ films increase in thickness more than 400% upon adsorption of lysozyme. These films contain a high concentration of free −COOH groups, and subsequent deprotonation of these groups at neutral pH likely contributes to increased protein binding. Lysozyme sorption stabilizes these films, as without lysozyme films deposited at pH 3.0 from 0.5 M NaCl desorb at neutral pH. Films deposited at pH 9.0 from 0.5 M NaCl are more stable and also bind large amounts of lysozyme. The high binding capacities of these films make them attractive for potential applications in protein isolation or immobilization of enzymes

Formation of High-Capacity Protein-Adsorbing Membranes through Simple Adsorption of Poly(acrylic acid)-Containing Films at Low pH

Layer-by-layer polyelectrolyte adsorption is a simple, convenient method for introducing ion-exchange sites in porous membranes. This study demonstrates that adsorption of poly­(acrylic acid) (PAA)-containing films at pH 3 rather than pH 5 increases the protein-binding capacity of such polyelectrolyte-modified membranes 3–6-fold. The low adsorption pH generates a high density of −COOH groups that function as either ion-exchange sites or points for covalent immobilization of metal-ion complexes that selectively bind tagged proteins. When functionalized with nitrilotriacetate (NTA)–Ni²⁺ complexes, membranes containing PAA/polyethylenimine (PEI)/PAA films bind 93 mg of histidine₆-tagged (His-tagged) ubiquitin per cm³ of membrane. Additionally these membranes isolate His-tagged COP9 signalosome complex subunit 8 from cell extracts and show >90% recovery of His-tagged ubiquitin. Although modification with polyelectrolyte films occurs by simply passing polyelectrolyte solutions through the membrane for as little as 5 min, with low-pH deposition the protein binding capacities of such membranes are as high as for membranes modified with polymer brushes and 2–3-fold higher than for commercially available immobilized metal affinity chromatography (IMAC) resins. Moreover, the buffer permeabilities of polyelectrolyte-modified membranes that bind His-tagged protein are ∼30% of the corresponding permeabilities of unmodified membranes, so protein capture can occur rapidly with low-pressure drops. Even at a solution linear velocity of 570 cm/h, membranes modified with PAA/PEI/PAA exhibit a lysozyme dynamic binding capacity (capacity at 10% breakthrough) of ∼40 mg/cm³. Preliminary studies suggest that these membranes are stable under depyrogenation conditions (1 M NaOH)