Recognition based separation of HIV-Tat protein using avidin–biotin interaction in modified microfiltration membranes

Abstract

Recognition based separation using modified microfiltration membranes provides an efficient and cost-effective alternative to conventional column chromatography for the separation and purification of a specific protein from mixture of proteins. In this study, Tat protein, which has been proposed as the specific target for AIDS vaccine, was separated and purified from a complex mixture of proteins, known as bacterial lysate (BL) using avidin–biotin interaction in 4-stack microfiltration membranes system. It was established by SDS-PAGE and Western Blot analysis that membrane based process recovered more pure form of Tat compared to conventional packed-bead column chromatography. The critical factors involved in the process, mainly, the accessibility of the covalently immobilized avidin sites by the biotinylated protein and the associated fouling of the membranes due to the permeation of proteins, were also studied. The accessibility of immobilized avidin sites in membrane was quantified by biotinylated solutions of different types and compositions. It was observed that permeation of proteins caused substantial fouling on the membrane matrix. The resistance offered by the protein layer and the approximate thickness of the protein layer were also quantified.