Efficient orthogonal integration of the bacteriophage ϕ29 DNA-portal connector protein in engineered lipid bilayers

The portal connector of bacteriophage viruses constitutes a robust molecular machine for DNA translocation. In this paper we propose an optimized reconstitution method for efficient orthogonal integration of native viral connectors into lipid bilayers, particularly of giant unilamellar vesicles. Our nanoengineering plan considers the hydrophilic connector protein of the bacteriophage virus ϕ29 integrated into a specifically engineered bilayer made of "hydrophylized" lipids. From the precise knowledge of the connector structure, the membrane chemistry was designed by tuning reactivity in the bilayer using specific functional lipids. We show details on the reconstitution methods and experimental evidence about the integration of the portal protein in the engineered membrane. The proposed route provides an efficient method for orthogonal integration of native viral connectors into lipid bilayers in conditions adequate for functional DNA translocation. This concept could be potentially exploited in advanced nanotechnological realizations, particularly for the integration of these powerful machines into giant lipid vesicles with the aim of building a cargo-device useful for gene delivery applications.