In this study, highly permeable ion-selective membranes are prepared via immobilization of polyelectrolyte multilayer networks within the inner pore structure of a microporous (pore size = 0.2 microm) support. Electrostatic layer-by-layer assembly is achieved through alternate adsorption of cationic and anionic polyelectrolytes under convective flow conditions. To initiate pore assembly, the first layer consists of covalently bound charged polypeptides (poly(L-glutamic acid) (PLGA) or poly(L-lysine) (PLL)) establishing a charged support for subsequent adsorption. Nonstoichiometric immobilization of charged multilayers within a confined pore geometry leads to an enhanced volume density of ionizable groups in the membrane phase. This overall increase in the effective charge density allows for Donnan exclusion of ionic species (especially divalent co-ions) using microporous materials characterized by permeability values that exceed conventional membrane processes. Multilayer assemblies are fabricated using both PLGA/PLL and synthetic polyelectrolytes (poly(styrenesulfonate)/poly(allylamine)) in an attempt to compare the level of adsorption and separation properties of the resulting materials. The role of salt concentration in the carrier solvent on overall polyelectrolyte adsorption was examined to determine its effect on both solute (Cl-, SO4(2-), As(V)) and water transport. Constriction of the pore size induced by multilayer propagation was monitored through permeability measurements and dextran rejection studies at each stage of the deposition process.

This work deals with the separation of Tat protein from a complex fermentation broth using an affinity membrane system. Tat is a regulatory protein that is critical for HIV-1 replication and thus a potential candidate for vaccine and drug development. Furthermore, Tat can facilitate transport of exogenous molecules across cell membranes and is implicated in pathogenesis of HIV dementia. Affinity membranes were prepared through coupling of avidin within a 4-stack membrane construct. Tat (naturally biotinylated) accessibility in the bacterial lysate feed was influenced by the presence of RNAse, protein concentration, and ionic strength. Enhanced accessibility translated to a marked increase in the overall product yield per pass. The purity of the membrane-isolated Tat was compared to that prepared via packed column chromatography through SDS-PAGE, Western blot, activity assay, and neurotoxicity studies. Tat protein produced via membrane separation yielded primarily monomeric forms of the oligopeptide sequence, whereas column chromatography produced predominately polymeric forms of Tat. These differences resulted in changes in the neurotoxicity and cellular uptake of the two preparations.

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