Designed multi-domain protein as a carrier of nucleic acids into cells

Abstract

Protein-based nucleic acid carriers offer attractive possibilities to enhance in vitro and in vivo gene delivery to combat diseases. A multi-domain fusion protein, namely TAT-NLS-Mu, designated as TNM, has been designed, cloned, heterologously expressed in E. coli and purified to homogeneity by affinity chromatography. The recombinant chimera TNM harbors three epitopes, a cell-penetrating (TAT) domain, a nuclear localization domain comprising of three nuclear localization sequence (NLS) motifs in tandem and a DNA-binding (Mu) domain. Complexes prepared by combining plasmid DNA with TNM (DP) transfect MCF-7, COS, CHO and HepG2 cells. Ternary complexes prepared with DNA, protein and cationic lipid (DPL) resulted in ~ 5–7 fold enhancement in reporter gene expression over the DP alone. Treatment of cells with chloroquine during transfection, with DP complexes, resulted in remarkable increases in reporter gene expression suggesting the involvement of endosomal compartments in the uptake process. Interestingly, DPL prepared with Lipofectin™ or 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) exhibited enhanced transfection in the presence of serum in MCF-7and HepG2 cells. Microinjection of DP complexes, with and without NLS sequence, into the cytoplasm and nucleus of smooth muscle cells (SMC) indicated that the presence of NLS sequence in protein carrier significantly enhanced transgene expression. Together the data suggest that modular design of proteins is a promising method to develop gene delivery carriers and also the role of NLS epitopes in mediating nuclear transfer of DNA complexes into various cell types.