Advancing Archaic Methodologies of Reconstituting Portal Proteins from Bacteriophages into Lipid Bilayer Membranes

The lipid bilayer membrane technology is vastly useful in explaining structural function of channel proteins, in the development of nanopore sensing technology as well as ultrafast DNA sequencing technology. In 2009, the portal protein, GP10, from bacteriophage phi29 was reported to be reconstituted into the planar bilayer membrane, which indicates the possibility of using the powerful technology to explore new applications of a large family of channel proteins from bacteriophages (Nature Nanotechnology, 2009, 4, 765-772). However, this utility comes at a great cost: For example, the portal protein reconstitution was time-consuming, unpredictable, and sporadic. Furthermore, the previously reported method was only limited to several hydrophilic mutant portal proteins and impossibly used as universal method to insert the portal proteins from other bacteriophages which are hydrophobic. In order to answer the questions, we proposed a method to form fusible vesicles reconstituted with portal proteins in the research. Using the vesicles, we were able to rapidly reconstitute portal proteins into planar bilayer membranes for the ionic channel current measurement. The time elapsed for 1st protein insertion was decreased by 14 folds. Furthermore, we could apply the methods to prepare vesicles reconstituted with hydrophobic proteins. Compared to previous methods, the new method proposed by our lab significantly extended the shelf-life of the proteoliposomes