(240f) Selective Separation of HIV-Tat Protein Using Functionalized Stacked Microfiltration Membranes: Enhancement of Flux and Recovery of Protein

Selective separation and purification of a particular protein from mixture of proteins is very critical for the downstream processing in biotechnology and pharmaceutical industries. Affinity chromatography using packed bead column is typically used for this type of separation. However, affinity based separation using functionalized microfiltration (MF) membranes is emerging as a cost effective and hydrodynamically favorable alternative to the conventional column chromatography. Affinity based separation in MF membranes is carried out by functionalizing the membrane pores with a suitable ligand and then selectively separating the target protein from the mixture using ligand-protein interaction. In this research work, avidin is used as the membrane immobilized ligand for the separation of biotin-tagged Tat protein from a complex mixture of proteins. Avidin-biotin interaction is chosen because of their very strong and selective affinity for each other. Stacked membranes system is used to increase the ligand loading and hence the protein recovery. TAT protein (M.W. 8335) is a regulatory protein of HIV-1 and is potentially an excellent target for AIDS related vaccines and drug development. Tat is genetically engineered to introduce a biotin structure while cloning in E. Coli vector. This biotin-tagged Tat is then separated from the fermentation broth (Bacterial Lysate, BL) containing other cellular proteins (>99%), cell debris and colloidal materials by permeating through avidin functionalized stacked MF membranes. For therapeutic usages Tat protein is functional in monomeric form only. The biggest challenge in Tat separation is to separate it in monomeric form as it tends to form polymers due to cysteine rich regions. It is established by SDS-PAGE, Western Blot, ELISA and Biotin analysis, that superior quality of monomeric Tat protein containing negligible biotin impurities is isolated by the membrane process compared to packed bead column chromatography. Two major concerns associated with this separation are the permeate flux and recovery of Tat protein. These can be improved if the unwanted high molecular weight impurities are removed from the system prior to the affinity separation. Thus a pre-filtration step, which helps in generating a dilute BL feed containing low molecular weight impurities along with Tat, is introduced before the affinity based membrane separation. Significant improvements in the flux and processing time of affinity separation are observed for the pre-filtered BL feed due to less fouling of the membranes system by the diluted protein mixture. The recovery of the Tat protein is also enhanced due to higher accessibility of the avidin sites by the biotinylated Tat. Some fundamental studies are also carried out on the fouling and accessibility of the covalently immobilized avidin sites by the biotinylated proteins. The transport of protein molecules through the membrane is described by a simple mathematical model developed by unsteady state mass balance. The fouling in the system is also described mathematically by considering non-specific adsorption of protein molecules on the pores and surface of the membranes.