Use of a kinesin-cro Fusion Protein as the Nanoshuttle to Transport Specific DNA

Kinesin motor proteins can hydrolyze ATP and unidirectionally transport specific cargo molecules along microtubule tracks (MTs). Engineering scientists interested in nanotechnology have used kinesin and MTs to build shuttles of 2 geometries. In the bead geometry, the kinesin shuttles transport cargos along the MTs. In the gliding geometry, cargos are carried by MTs, which glide on a kinesin lawn. To realize the goal of building in vitro protein shuttles with real utilities, the issue of how to allow the shuttles specifically, yet easily, to transport the designated cargos has not been completely resolved. Popular methods, such as non-specific bead attachments, or biotinylation of tubulin subunits by covalent tagging with reactive chemicals, have limitations. In this study, we focus on developing a bead geometry protein shuttle to transport specific DNA. Because numerous DNA-binding proteins must form homodimers to bind DNA, we created kinesin fusion proteins KIF5-cro proteins. These proteins were made by genetically fusing the tail of a truncated kinesin with the cro protein, which is a DNA-binding protein derived from the λ bacteriophage. We showed that the resultant kinesin fusion proteins transport DNA with specificity and high affinity (Kd 60 nM). This study provides a discussion on the plausible approaches of applying the recombinant DNA technology to the shuttles of the gliding geometry.