Selective separation of HIV-Tat protein using functionalized stacked microfiltration membranes: Enhancement of flux and recovery of protein

Affinity separation using functionalized microfiltration (MF) membranes provides a hydrodynamically favorable and cost-effective alternative to conventional column chromatography for the separation and purification of a specific protein from mixture of proteins. This type of separation in MF membranes is carried out by functionalizing the membrane with a suitable ligand containing specific interaction sites, and then selectively separating the target protein from mixture under convective flow. 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 is a regulatory protein of HIV-1 and is potentially an excellent target for AIDS related vaccines and drug development. Tat was genetically engineered to introduce a biotin structure by bridging with a fusion protein while cloning in E. Coli vector. This biotin-tagged Tat was then separated from the fermentation broth (Bacterial Lysate, BL) containing other cellular proteins (>97 wt %) and impurities (cell debris, etc.) by permeating through avidin functionalized stacked MF membranes. For further medical uses Tat protein is functional in monomeric form only. The biggest challenge in Tat separation was to separate it in monomeric form as it tends to form polymers due to cysteine rich regions. It also binds with other cellular proteins.